Month: March 2025

Moreover, the Environmental Protection Agency of the United States ruled that MeJA was exempted from the requirement of a tolerance for residues in or on all food commodities when applied pre-harvest , a ruling that could facilitate the development of jasmonates as active ingredients in agrichemicals. A screening trial confirmed that MeJA and coronatine, a jasmonate mimic, also induce abscission in grape. The physiological basis for jasmonate-induced fruit abscission appears to involve ethylene. The exogenous application of jasmonates stimulates ethylene production in several fruits, including apple , orange, grape, strawberry , and tomato. The work carried out on orange and grape showed that the application of MeJA stimulated ethylene production by the fruit which was followed by fruit abscission. Malladi et al. provided indirect evidence that MeJA stimulates the abscission of blueberry fruits at least partly via ethylene action, as the co-application of MeJA with aminoethoxyvinylglycine , an ethylene biosynthesis inhibitor, attenuated MeJA effects on abscission. However, MeJA still induced some abscission in blueberry, even when co-applied with AVG which suggests that MeJA may initiate some abscission processes independently of ethylene. Moreover,grape berries treated with MeJA or 1-aminocyclopropane-1-carboxylic acid , the direct precursor of ethylene, produced similar levels of ethylene in the first 2 days after treatment , but thereafter, berries treated with MeJA produced less ethylene than berries treated with ACC, even though the MeJA-treated grapes generally had lower FDF, greater abscission, drainage gutter and a higher proportion of dry stem scars than the grapes treated with ACC. Together, these findings suggest that ethylene and jasmonic acid can promote fruit abscission via independent pathways and interact to promote abscission.

The potential for synergistic effects has sustained interest in research on the coapplication of jasmonates and ethylene-promoting compounds. This is especially important because of the relatively high dosages of MeJA needed for consistent efficacy when applied alone and because MeJA is much more expensive than ethephon. Because of the high rate of ethephon needed for abscission activity, the short time between abscission zone activation and fruit drop, and concerns about ethephon residues, alternatives to ethephon are desired. 1-Aminocyclopropane-1-carboxylic acid is not particularly effective at stimulating abscission on its own, but the co-application of MeJA with ACC improved efficacy in such a way that lower dosages of MeJA could be used. Recent improvements in jasmonic acid biosynthesis have the potential to make JA and its metabolite MeJA more available and affordable than they are now. The effects of JA on the abscission of fleshy fruits has not been tested, and the relative efficacy of JA versus MeJA with respect to grape berry abscission is unknown. Therefore, two studies were conducted to compare the efficacy of JA and MeJA at inducing abscission of Thompson Seedless grapes and to determine if JA interacts with ACC to promote abscission.Methyl jasmonate at 2 mM was ineffective, whereas 4 mM and 8 mM MeJA were equally effective at inducing preharvest abscission, reducing fruit detachment force , and increasing the proportion of detached berries with dry stem scars . Jasmonic acid was as effective or more effective than MeJA at inducing the abscission of Thompson Seedless grapes. Compared with MeJA, JA induced a similar or higher preharvest berry abscission, a similar or lower fruit detachment force, and a similar or higher percentage of detached berries with dry stem scars after treatment with JA, versus MeJA . The most effective treatment overall was 4 mM JA, which induced the highest level of preharvest abscission and percentage of detached berries with dry stem scars .

Grapes that were treated with 4 mM JA also measured among the lowest FDF values .MeJA can stimulate the abscission of many fruits and endogenous JA is known to promote abscission of floral organs and fruits, but the data presented here may be the first report of an exogenous application of JA stimulating the abscission of a mature fruit. Moreover, JA appears to be at least as effective, and possibly more effective, than MeJA at stimulating grape berry abscission. Interestingly, this is in contrast with a recent study that showed MeJA was more effective than JA at inducing abscission in lupine flowers. Improved efficacy at lower dosages could facilitate the commercial development of jasmonate-based abscission agents for grapes and other fleshy fruits because these natural products are expensive. In the first study, 4 mM JA was more effective than 8 mM JA; however, the opposite appeared to be the case in the second study. Therefore, additional research is needed to clarify the lowest dosage of JA that is consistently effective. The general range of effective dosages agrees with previous research that used MeJA as the active ingredient. Abscission zone activation reduces FDF and promotes the development of dry stem scars, both of which could help minimize picking damage and possibly improve the quality of destemmed table grapes. However, the final stage of AZ activation is undesirable unless catchment systems can be employed. Previous studies with MeJA suggested that harvest should occur within 3 days after treatment. Jasmonic acid also stimulates rapid abscission zone activation, with 14 to 25% abscission observed 2 days after treatment, and 52% to 60% by 3 DAT.

Without catchment systems, it may be necessary to harvest treated fruit within 2 days to avoid excess crop loss. This could be logistically difficult, and data are lacking to determine whether the abscission zone would be sufficiently developed by 1 or 2 DAT to provide the potential quality benefits that are desired from abscission agents. Another outcome that needs to be determined is whether the abscission zone could be activated preharvest and develop during postharvest storage. If so, this could be a way to achieve fruit quality benefits for stemless table grapes while minimizing the risk of preharvest fruit drop. Lavee demonstrated that preharvest applications of plant growth regulators can affect the postharvest abscission of grapes. Specifically, the preharvest application of 1- naphthaleneacetic acid and some other synthetic auxins reduced the postharvest abscission of “Muscat of Alexandria” that were held at room temperature for three days before entering cold storage. However, the abscission of grapes that were placed into cold storage immediately after picking was greatly suppressed, regardless of whether the grapes were pretreated with auxins or not. This suggests that the effect of plant growth regulators on postharvest abscission may depend on postharvest storage conditions. If abscission zone activation and development could both occur postharvest, it may be possible to treat the fruit in a packing house which should enable more efficient application of active ingredients, in addition to preventing crop loss due to preharvest abscission. The results of the second experiment also demonstrate a benefit in applying ACC with JA, as previously shown with MeJA. The co-application of 500 ppm ACC was sufficient to make 4 mM JA as effective as 8 mM JA in promoting dry stem scars, one of the most important treatment effects with respect to the quality of detached grapes. Because 500 ppm was the lowest dosage of ACC tested, it is unknown if a lower dosage might be as effective, but this should be tested in future research since the 500 ppm and 1000 ppm ACC treatments had similar interaction effects with JA. Likewise, a lower dosage of JA could be effective, especially if combined with ACC. Thus, additional work should be carried out to determine if combination treatments could enable lower dosages of JA and ACC to be reliably effective. In conclusion, plastic gutter the exogenous application of JA activates the pedicel-fruit abscission zone of Thompson Seedless grapes, rapidly reducing FDF, increasing the proportion of berries with dry stem scars after detachment, and leading to significant preharvest abscission within 2 days. Treatment effects require more than 2 mM and less than 8 mM JA if applied alone, but possibly less if co-applied with ACC. Additional work is needed to determine if harvest within 2 days after treatment is sufficient to reduce FDF and increase dry stem scar incidence while reducing preharvest abscission. Work should also be carried out to determine if preharvest or postharvest treatments are effective at inducing abscission postharvest.The experiments were conducted in September 2020 with own-rooted Vitis vinifera cv. Thompson Seedless grapevines and supported by an overhead-arbor trellis in a vineyard at the University of California Kearney Agricultural Research and Extension Center, in Parlier, CA. The vines were planted in 1995, trained to quadrilateral-cordons, and canepruned, leaving approximately 6 canes per vine, and 15 nodes per cane. Vines were spaced approximately 1.83 m within rows, and 3.65 m between rows which were oriented east to west.

All vines were subjected to cultural practices considered normal and ordinary for dry-on-vine raisin grapes in the San Joaquin Valley, except that the canes were not severed, and raisins were not made. Each of the two experiments used some methods comparable to those employed in a previous study. Clusters on individual vines were considered treatment replicates and each vine was considered a block. Since there were two experiments, each replicated six times, two groups of six adjacent vines were identified, with one group assigned to the first experiment, and the second group to the second experiment. There were seven treatments in the first experiment: an untreated control; 2 mM, 4 mM, and 8 mM MeJA ; and 2 mM, 4 mM, and 8 mM JA . Unique tags were made for each replicate and treatment and placed in different bags, according to their block. Available clusters were randomly assigned to each treatment by pulling the tags out of a bag and tying them to the peduncle of each cluster. The morning after labelling each cluster, solutions were prepared with the proper amount of MeJA or JA in water, with 0.05% of Latron-B1956 spreader-sticker . Control treatments consisted of water with Latron-B1956. Each solution was placed into a spray bottle , agitated well, and then applied to an appropriately labelled cluster until runoff occurred. Polyethylene shields were used to prevent overspray from contacting other clusters. After the clusters dried, they were enclosed in polypropylene mesh bags to catch any berries at risk of abscission. The bags had a re-sealable flap at the bottom from which abscised berries could be collected and weighed. In the first experiment the berries were collected once, 3 days after treatment. The abscised berries were weighed and discarded, and then each cluster was harvested and taken to a laboratory in their mesh bags where FDF and dry stem scar measurements were made. At the laboratory, clusters were gently removed from their bags, and small shears were used to sever 10 berries from the top, middle, and bottom part of each cluster, retaining the pedicel and a short section of rachis with each berry. Each berry was then placed in a jig attached to a force gauge , and force parallel to the fruit axis was applied to the rachis until it detached from the berry and peak force was recorded. After each berry was detached, the stem end was observed and assigned to one of two classes: dry or wet stem scar. The remaining berries on each cluster were then removed, combined with the ten used for FDF and dry stem scar measurements, and weighed. The total weight of each cluster was determined by adding the weight of any berries that had abscised prior to harvest, and the percentage of preharvest fruit drop was calculated based on the cumulative weight of the abscised berries and the weight of the berries remaining at harvest. The second experiment had nine treatment combinations: three levels of JA in a factorial design. The ACC was provided by Valent BioSciences . Cluster selection, solution preparation , treatment application, and bagging, were similar to the procedures employed in the first experiment. In the second experiment, cumulative preharvest abscission was determined daily, by weight. After 3 DAT, all the clusters were taken to the laboratory for determination of FDF, dry stem scar development, and cluster weight determination. All data were subjected to analysis of variance using the GLM procedure of SAS . In the first experiment, a randomized complete block design was used, with seven treatments. Means were compared by Duncan’s new multiple range test .

Our study differs from prior peach transcriptomic analyses in two ways. First, we are using samples from pools of genetically related siblings with contrasting sensitivity to chilling injury subjected or not to cold storage. Thus we expect to reveal genes whose expression patterns are linked to the different cold sensitivity, while leveraging transcript differences associated with other phenotypic traits, as it would be the case when comparing only two peach cultivars that have different chilling susceptibilities in addition to other phenotypic differences. Second, by medium throughput qRT PCR we extended our microarray results derived from the comparison of the contrasting pools to a relatively large number of 15 individual lines from the same population differing in the mealiness sensibility and the gene expression results of the selected genes were consistent with their individual sensitivity level.Orthologs of several transcription factors found upregulated similarly in S and LS cold-treated fruits have been previously reported as being up-regulated during cold acclimation in Arabidopsis and some of them also were described as belonging to a given cold acclimation regulon. This suggests the activation of a cold response program in peach fruits in part similar to those described for Arabidopsis cold acclimation. Despite observing similarities some genes exhibited an opposite trend compared to Arabidopsis which may partially reflect the sensitive character of peach fruit to cold . Several studies have associated cold tolerance and cold acclimation the transcriptional activation of genes encoding heat-shock proteins , chaperonins, LEA proteins, antioxidant/scavenging systems and related to protein synthesis.

Genes in these functional categories were generally down-regulated by cold storage in both LS and S fruits, square plastic planter what correlates well with their sensitivity to cold. Further, the orthologs of HSF4B and HSP21 were up-regulated peach fruits, whilst were down-regulated in Arabidopsis. This is particularly interesting as these genes are highly up-regulated in Arabidopsis chilling sensitive mutants upon chilling treatment. It should be noted that we are comparing the transcriptomes of different species and tissues at various physiological and growth stages, and it is likely that some differences in strategies to cope with exposure to low temperatures operate in each case. The basic question is: why do LS PopGG siblings tolerate better cold storage than S? Our results indicate that during cold storage fruits LS maintain higher levels of expression for a series of components of the antioxidant system, structure maintenance proteins and protein synthesis at least during the first week of storage . In addition, the orthologs of some TF with a higher expression levels in tolerant peach fruits have been reported to be upregulated by cold and/or other biotic or abiotic stresses in Arabidopsis . In this sense, our data indicated that the peach orthologs for genes in ICE1, CBF and HOS9 regulons may be implicated in the tolerance of fruits LS. The central role played by the ICE1-CBF cold response pathway in cold acclimation and cold tolerance is well-established in plants and has been demonstrated to exist in a wide range of plants, although, there are differences in the regulation or the size of their CBF regulons. The existence of ICE-CBF pathway has been also confirmed in fruits. Further, LeCBF1 expression levels correlates positively with cold tolerance in tomato fruits. We found that genes in the regulons ICE1and CBF were the most contributing to discriminate samples S from LS, and/or to separate samples that will become mealy, or not .

Moreover, PCA analysis identified CBF1 as the second gene that contribute the most to separate the S and LS series and qRT PCR analysis showed that the expression levels of CBF1 correlate well with the tolerance/ sensitivity of the individual pop-DG siblings . Thus, confirming ICE-CBF as important actors in the differential response to chilling between peaches S and LS. In the case of the genes in regulon HOS9 our results suggest that it is more likely related with the ability to up-regulate or to maintain similar expression levels to those observed in M fruits . Zhu et al. concluded that HOS9 must be important for both the constitutive expression and cold-induced expression of the genes that may be required for full tolerance to freezing stress. These results are consistent with peaches having the basic components of a cold response pathway, but additional studies will be required to elucidate their size and how they are regulated. In normal commercial fruit operations cold storage, involves also complete darkness. Gene by gene comparisons has revealed that around 3% of our cold regulated genes in peaches could be related to darkness . Moreover, we identify some genes whose orthologs have been described in the regulation or in response to light . Several, light sinaling elements among which were GI, DFL2, PHYA and FYPP3 were repressed by cold storage in both LS and S , consistently with the storage in darkness conditions. In addition, genes differentially expressed between fruits S and T include a number of regulators involved in light response that indicates we should take into account this factor as contributing the differential response observed in peach fruits. In Arabidopsis light is required for cold induction of several genes involved in cold acclimation, including CBFs and some light signaling mutants have impaired cold acclimation. Thus the differential response to cold storage of fruits S and LS probably have to do fruits’ ability to deal with cold and darkness. However, further experiments are required to determine in more detail the nature of the interaction between the cold and the darkness during storage.Despite no visible mealiness symptoms are observed during cold storage, the BSGA indicated dramatic changes in the peach transcriptome in response to the exposure to mealiness-inducing temperatures in a manner that these changes could be useful to predict future mealiness development .

We propose the transcript differences observed while in the cold might underlie the molecular basis of a mealiness phenotype which is still undetectable, but will be fully developed later during shelf life. This is in agreement with previous reports of the cold induction of specific target genes that are associated with the mealiness disorder. Surprisingly, our results showed that cell wall is not found among enriched categories in none of the clusters/comparisons performed on cold stored samples, suggesting that although specific changes in cell wall remodeling transcript are detected most of the changes would probably occur during shelf life. Our results reveal also that transport and signaling elements presented higher levels in S fruits, which in some cases, correlated well with the eventual mealiness phenotype. We found the orthologs of genes described as positive regulators of ABA signaling and/or osmotic stress and transporters related to Na+ and K+, sugar and nitrate homeostasis among genes high expressed in fruits S This suggests that fruits S during cold storage undergo some sort of dehydration or osmotic adjustment. It has been proposed that during cold storage, before mealiness is manifested, pectin depolymerisation but not deesterification is inhibited, square plastic plant pot what may lead to the formation of gel-forming pectins that traps free water from the surrounding tissue. As no significant differences in global water content are found between LS and S fruits it is likely that water is being lost from the cell to be trapped on the pectins of the cell wall, which still would be sensed as loss of internal water by the cell. Among genes with higher expression in sensitive fruits we identified components of auxin and ethylene signaling cascades as well the orthologs of genes involved in the biosynthesis of ABA, auxin and ethylene . We must highlight the large list of genes related to auxins among with were positive regulators of auxin responses and transporter locations . In addition, among the genes high expressed in the fruits LS at one week there were the orthologs of genes such as HAB1, PP2CA/AHG3, SAD1 and ERD15 ,which have all been described as negative regulators of ABA signaling, and IAA17/AUX3, proposed to be a negative regulator in auxin and ABA signaling. Ethylene and auxins has been described in the regulation of the ripening program of peach fruits and their involvement in the cold response has been described for Arabidopsis, tomato, apple and peach. Our results indicate that part of the ripening program probably continues during cold storage in sensitive fruits . Hence, we could expect that interactions between cold and hormones controlling the peach ripening program, which are differential between fruits S and T, impact the way fruits respond to cold and ripen afterwards during shelf life. Because the activity of most of these genes is mainly determined at post-trasncriptional level reviewed in [90], it is not possible from expression data only to infer the role of these genes during cold storage. However from our data it is clear that all three hormones may play a role in regulating the differential response of peach fruits to cold and they seem operate in association with dehydration/osmotic stress. In support of that, the orthologs of many of hormone related genes higher expressed in CS1-S fruits have been described previously either in relation to drought and osmotic stress . For example, the orthologs of SKIP, BRM and ERD1 mediate the responses or are induced by ABA, salinity and dehydration stress; CPL2 modulates auxin responses, plant growth and osmotic stress and EIN2 has been described to be an important cross-link node for the interaction of ethylene, ABA and plant response to abiotic stress. We cannot rule out that the ‘‘sensitivity’’ program is the consequence or the cause of low levels ICE1-CBF regulons. It is possible that the up-regulation of a set of common genes concomitantly with low CBF levels triggers this program. It is also feasible that among CS1 S.LS there are genes which negatively regulate the CBF response. To support this, EIN2 has been described as a negative regulator of plant response to freezing stress by negatively regulating the expression of CBF1-3 and its target genes; interestingly, CBF genes have been found to be directly repressed by IAA. Finally, it may also be possible that this program is activated to compensate efficient acclimation during cold storage. It has been described that hos9 mutants hyperactivate some cold-regulated genes through a compensating response to their increased cold sensitivity.At the mature stage specific differences at the gene expression level between the pools of fruits S and T already exist . Although our approach used pools of fruits in accordance to how they respond to cold storage, therefore minimizing differences in other aspects between genotypes, we can’t dismiss the possibility that these differences have nothing to do with adaptation to cold. Preformed mechanisms have been described in both biotic and abiotic stress tolerance and we previously identified a subset of genes differentially expressed at harvest that correlate well with CI. Cell wall metabolism has been extensively related to mealiness in peach fruits, and it has been reported that endopolygalacturonase plays a qualitative role in the mealiness expression. Our results indicate that the composition of the cell wall at harvest could play a role in the tolerance or sensitivity of peach fruits to withstand cold storage. This is in agreement with previous results. In addition the type of functional categories for the differentially expressed genes at the stage M, and the fact that most of these genes continue to show these differences during cold storage , suggest the possibility that a pre-programmed tolerance/sensitivity mechanism can be partly established previously to cold. Among the highly expressed genes in fruits LS at the mature stage, we found orthologs of genes such as CHS/TT4 and GST12/TT19 , which have been described being essential for anthocyanin and proanthocyanin accumulation. Anthocyanins have been related with browning in peaches. However, no significant differences in browning, bleeding nor in ppLDOX expression were observed between our pools. It is suggested that AtTT19 functions as a carrier to transport proanthocyanin precursors to the tonoplast to be later secreted and linked to cell wall polysaccharides. Binding that depends on the composition of the proanthocyanin. The tt19 mutation leads to the formation of aberrant PA derivatives. Thus is possible that differences in TT19 have to do with cell wall composition and chilling sensitivity. Further experiments are required to test this hypothesis. In addition, flavonoids act as negative regulators of auxin transport.

The model suggests that in colder areas, the observed late-season buildup of the fly results from low winter survival and the movement of adults from areas with phenologically earlier populations.Berry curvature is a key to understand novel physical phenomena such as anomalous Hall effect, chiral anomaly, topological Hall effect, and spin-valley Hall effect. Moreover, the BC classifies the topology of a solid via a topological number that predicts the presence of protected states at its boundary. The response of a system governed by the BC is constrained by the Onsager relation in the linear order. This stringent constraint, however, is no longer valid in the high-order responses that are proportional to the second-order or even higher orders of the driving field. The integration over higher-order fields gives a nonlinear response of the system, contributing to the optical and transport responses . This finding not only provides a methodology to explore the momentum texture of the BC of a system, but also paves a way to utilize the response even though the linear order is vanishingly weak or when a large driving field makes the higher-order response exceed the linear order. To make use of the higher-order response has great potential for applications in rectification devices , photosensitive devices , and photovoltaic devices that potentially overcome the quantum efficiency limit.Recent studies on the higher-order response have invited nonmagnetic materials with broken centrosymmetry as a new member of the Hall effect family, so-called nonlinear Hall effect , characterized by a quadratic behavior of the Hall voltage with second harmonic frequencies in the presence of a perpendicular AC driving current. In the absence of the linear Hall effect due to time reversal symmetry, 25 liter plant pot the lowest-order Hall current is driven by the Berry curvature dipole.

Since the response is proportional to the gradient of the BC, tilted anticrossing bands and Weyl points are predicted to exhibit strong BCD that can generate a nonlinear Hall angle close to 90 degrees. As a result, the momentum-dependent texture of the BC based on the electronic structures is essential to understand the NLHE in which the evolution of BC momentum texture by lattice strain, interlayer twisting, and external electric fields can lead to NLHE-based device applications. Nevertheless, while the majority of studies on the NLHE have predominantly focused on exploring its transport properties, applications, and theoretical simulations, the direct experimental confirmation and comprehensive understanding of the BCD based on electronic structures have not yet been fully attained. This achievement would provide crucial insights into the underlying mechanism and controllability of the BCD. In this paper, we report the room temperature NLHE in NbIrTe4 thin flakes, which exhibit a frequency-doubled Hall conductivity proportional to the square of the driving current. We also demonstrate that the sign change in the NLHE at 150 K is induced by the sign change of the BCD because of the chemical potential shift at high temperatures. It is unambiguously evidenced by direct observation of a chemical potential shift in the temperature-dependent band dispersion using angle-resolved photo emission spectroscopy and calculated BCDs. Investigation of the electronic structures using ARPES and density functional theory also indicates that the main contributor of BCDs is a partial occupation of spin–orbit split bands. Our findings provide important insights into the momentum texture of the Berry curvature and into controlling the Berry curvature dipole hosting the NLHE, which can be utilized for NLHE-based devices.

It is often asserted and then assumed that fundamental research from model systems, such as Arabidopsis, is valuable because this knowledge would be utilized later to elucidate practical aspects of plant function in species of agronomic importance. This has been successful in like organs and developmental processes. For example, a regulatory module involved in photoperiod control of flowering has been elucidated through extensive research in Arabidopsis , and more recently this same mechanism has been shown to control flower time in Populus trichocarpa . Unexpectedly, Bohlenius et al. revealed that the same regulatory module is involved in the control of bud dormancy. This illustrates that components of regulatory mechanisms can be conserved across disparate developmental processes that share commonalities; a dependence on daylength and action in the shoot apex in the case of flowering and bud dormancy. Less is known about the utility of extending knowledge to a class of organ not even present in a given model species. The onset of ripening marks a transition in fruit development leading to changes in physiology involving sugar metabolism, softening, and color development. Most understanding of this process arises from work with climacteric fruits in which the control of ripening is predominately by ethylene. However, there are many fruits of economic importance that are nonclimacteric, such as grape, strawberry, and citrus, where the processes controlling the onset of ripening are still poorly understood. Study has traditionally focused on ethylene in climacteric fruits and on other hormones such as sugar and abscisic acid in non-climacteric fruits. However, there are numerous studies implicating ethylene in the ripening of non-climacteric fruits , and conversely, sugar and ABA in the ripening of climacteric fruits . In addition, other hormones have been show to influence the ripening processes such as auxin and brassinosteroids .

Fruit ripening in general involves the integration of multiple hormone signals. Climacteric and non-climacteric fruits are not delimited by phylogeny and both exist even among closely related species . This suggests that ripening in all fleshy fruit may have a common foundation. Sugar and ABA function as signals in many aspects of plant development and there are numerous lines of evidence correlating increases in sugar and ABA with the onset of ripening in grape . In field-grown berries, exogenous ABA increases anthocyanin accumulation , and in our own work berries fail to synthesize anthocyanins when sugar import into the berry is disrupted via phloem girdling prior to the onset of ripening . Finally, water deficits also result in increases in anthocyanin accumulation , and have been shown to increase ABA accumulation at the onset of ripening . Many studies investigating the role of sugar and ABA in ripening have focused on color development. In viticulture, the onset of ripening is traditionally referred to as veraison and is defined by the initiation of color development. More contemporary genomic studies demonstrate that color can act as a proxy for developmental stage in that individual grape berries of like color have similar global gene expression patterns . A previous study in our laboratory found that water deficits advanced the onset of color development, but sugar and anthocyanin accumulation remained closely linked . Furthermore, the induction of core flavonoid and anthocyanin-specific genes reflected this advancement as well . Therefore, in the current study, water deficit is used as a means to manipulate the timing of the onset of ripening in the field. There are few examples of studies focused on identifying the molecular mechanisms responsible for sugar and ABA action in grape, although several recent studies have demonstrated roles for sugar and ABA in controlling sugar metabolism. ABA stimulates acid invertase activity , and expression of the grape hexose transporter VvHT1 is regulated by both ABA and hexose . Two other recent studies have focused on core sugar and ABA-signaling components. A calcium-dependent protein kinase is regulated by ABA during ripening , and a study in grape cell culture provides evidence that sugar induced increases in anthocyanin accumulation are dependent on calcium, calmodulin, and kinase activity . In this study, we begin to examine the hypothesis that orthologous sugar and ABA-signaling components, black plastic plant pots characterized across such diverse processes as seed dormancy and stomatal control, function similarly in the control of the onset of ripening in fleshy fruit. This study employs a comprehensive approach in identifying novel sugar and ABA-signaling candidates in grape.

Ten gene families, containing sugar and ABA-signaling components elucidated in model systems, were considered including; the putative sucrose sensor SUT2, core G-protein signaling components GPA1 and RGS1, hexokinases , PP2C protein phosphatases, Snf1-related kinases , and the sugar-related WRKY, and ABA-related homeodomain–leucine zipper, or homeobox , ABRE-binding factor , and AP2 transcription factors . Their orthologous families in grape were identified, and the expression of individual genes were characterized in control- and deficit-irrigated, field-grown Cabernet Sauvignon. The onset of ripening was induced in cultured immature berries with sugar and ABA treatments. Finally, the effect of exogenous sugar and ABA on the expression of several key orthologs was investigated in berry culture.The field experiment was the same as presented in Castellarin et al. . In short, experiments were conducted during 2006 in a commercial vineyard of R.H. Phillips Winery using Vitis vinifera ‘‘Cabernet Sauvignon’’ clone 337 grafted onto rootstock 140R. Two irrigation treatments were established. Irrigation was applied to control vines in order to maintain midday leaf water potential between -0.9 and -1.2 MPa. Irrigation was cut off to early deficit vines at fruit set until 77 days after anthesis ; then vines were irrigated at the C rate till 133 DAA. Plant water status was monitored weekly by measuring midday leaf water potential according to Matthews et al. . Each treatment was replicated four times in 0.5-ha plots dispersed over a 36-ha vineyard according to a randomized complete block design. Samples were collected randomly from ten vines located in the central row of the plots. Berries were sampled six times between 27 June and 5 October , when the grapes were harvested. Twenty berries were collected from each plot for use in studies of gene expression. During the onset of ripening, samples of green and red berries were collected separately from the same clusters and analyzed separately.Cabernet Sauvignon clusters were collected from fieldgrown vines at 61 DAA. Clusters were surfaced sterilized for 10 s in a 70% ethanol solution, 10 min in a 1% bleach solution, and then rinsed three times with sterilized water. Berries were removed from the clusters by cutting the stem at the peduncle level, and the peduncle was removed from the rest of the fruit with a scalpel. Berries were weighed and individually cultured for 23 days in 20 mL glass vials filled with 7 mL of six different 1% agarose media 2% sucrose, 10% sucrose, 200 lM ABA alone, 2% sucrose 200 lM ABA, 10% sucrose 10 lM ABA, 10% sucrose 200 lM ABA. The 2 and 10% sucrose concentrations used in this study approximate sugar concentrations of berries prior to and at , the onset of ripening in the field . Each treatment was replicated ten times. Berry color changes were visually analyzed twice, 10 days after T0 and at the end of the experiment. Elasticity was measured at T0 and at the end of the experiment. In short, berry elasticity was determined non-destructively using a custom-fabricated instrument, which measured force and displacement during berry compression. As described in Thomas et al. , the observed force/displacement relation was fit using SAS PROC NLIN to that expected for compression of a perfectly elastic sphere . 23 days after T0, berries were collected from the vials, weighed, and stored at -80 C till the tissues were processed for the RNA extractions. Three berries from the 2% sucrose, 10% sucrose, 10% sucrose 10 lM ABA, 10% sucrose 200 lM ABA treatments were peeled and total RNA was extracted from each individual skin following the procedure described above.Utilizing the sequenced grape genome , 67 genes were identified representing orthologs of sugar and ABA-related genes in 10 gene families . The genomic complexity of these families varied from small families with just one or few genes to large families made up of nearly 100 genes . Small gene families were comprehensively characterized for all grape orthologs, while only those subgroups containing genes specifically shown to be involved in sugar and ABA signaling were included for large gene families. Given the scope of this work, we focused primarily on those gene families encoding the following well characterized orthologs the Arabidopsis Class I HB transcription factors, the Arabidopsis ABA-insensitive mutants, abi1 and abi2, encoding PP2C protein phosphatases, and the WRKY transcription factors SUSIBA2, AtWRKY4, and AtWRKY34. Other gene families are summarized and referenced as supplemental material when pertinent. The homeodomain–leucine zipper, or homeobox transcription factors represent a medium-sized gene family with approximately 47 members in Arabidopsis.

The Chern number is just a property of a band and does not come with an energy scale, so there is no reason to expect to encounter Chern bands only at low temperatures. Indeed, bands with finite Chern numbers have been shown to support quantized Hall effects in graphene quantum Hall devices at room temperature and high magnetic fields, as illustrated in Fig. 8.5A,B. The energy scale in a Chern magnet is set by the band gap produced by magnetic interactions. So if we’d like to know what the maximum temperature at which we can expect to find Chern magnets is, we need to think about the energy scales of known magnets. Magnetism is an interaction-driven electronic phase, and interaction-driven phases almost always melt at sufficiently high temperatures. However, among interaction-driven electronic phases ferromagnetism is particularly stable. Many common transition metals, including iron, cobalt, and nickel, support ferromagnetism into the range 600-1200 K, and all of these have found applications in a variety of electronic technologies as a result. These are of course all three dimensional crystals, and Chern magnets are two dimensional crystals. So the next question we can ask is: do two dimensional magnets exist with Curie temperatures as high as room temperature? The answer turns out to be yes, as illustrated in Fig. 8.5C,D. This magnetic system appears not to be a Chern magnet, unfortunately, but the point is that there is nothing in particular stopping a Chern magnet with a Curie temperature above 300 K from existing. The first intrinsic two dimensional ferromagnets were discovered in 2017, so I think it’s safe to say that our field hasn’t yet come particularly close to identifying all possible two dimensional magnets. It’s hard to do an accurate accounting of all of the so-far discovered two dimensional magnets, drainage pot and it is certainly the case that many of these are are not Chern magnets.

But of the two dimensional magnets we have found, a surprisingly large fraction are intrinsic Chern magnets. We know of eight intrinsic Chern magnets stable in the absence of an applied magnetic field in the published literature so far. These are presented, along with a few of their basic properties, in Table 8.1.Over the course of my PhD, four nanoSQUID microscopes were proposed, and construction began in some form on all of them. By the time I left we had finished three of these microscopes. The first nanoSQUID microscope we completed was inserted into a bath of liquid helium and could operate at 4 K. The second nanoSQUID microscope had a pumped He-4 evaporative cooling pot, and could reach temperatures of 1.5 K. The tBLG/hBN Chern magnet transport measurements, the tBLG/hBN Chern magnet imaging measurements, and the AB-MoTe2/WeSe2 Chern magnet imaging measurements were all performed in this system. The third nanoSQUID microscope had a closed cycle He-3 sorption pump cooling system, and could reach 300 mK. The ABC trilayer orbital magnet imaging measurements were performed in this system. The fourth and final microscope remains under construction, and is designed to operate inside of a dilution refrigerator. Pictures of several of these microscopes are shown in Fig. 8.6. Acoustic isolation chambers and the 300 mK system are not shown. All nanoSQUIDs have liquid He-4 baths for primary stage cooling, and all are mounted on several thousand pound vibration isolation tables floating on air legs to protect the nanoSQUID sensors from mechanical and acoustic shocks close to the surface. The nanoSQUID sensor circuit is fairly simple, with only one important non-standard circuit element in it, other than the nanoSQUID itself of course. This is the series SQUID array amplifier. Current is forced into the nanoSQUID sensor in parallel with a shunt resistor of comparable resistance to the nanoSQUID sensor in the voltage state, which is generally a few Ohms.

Current through the nanoSQUID side of the circuit is inductively coupled to a series of identical SQUIDs. These SQUIDs in series generate a large voltage, which is detected at room temperature. Current is forced through a feedback coil to maintain constant flux through the SQUIDs in series. This allows the circuit to maintain sensitivity over a wide range of currents . This current amplification circuit has good current sensitivity and enormous dynamics range, easily able to accommodate the several hundred µA necessary to reach the critical current of the nanoSQUID sensor.There are a lot of things that make scanning probe microscopy tough relative to other techniques for performing microscopy. One particularly challenging issue is navigation of the sensor to the sample. Those experienced with optical imaging might be spoiled by a contrast mechanism that is sensitive to a ton of different phenomena- the nanoSQUID can only see local gradients in magnetic field and temperature, and those are rare unless you have intentionally built structures and devices that generate them for use in navigation. In particular, large thermal gradients and variations in local magnetic field aren’t general properties of surfaces, so it’s very easy to blunder a nanoSQUID sensor into a surface without ever seeing it coming! Experiments are thus much safer and more expedient if we can provide the nanoSQUID sensor with topographic feedback- i.e., some way of detecting surfaces without crashing into them and destroying the sensor. We did this using shear force microscopy, which is a form of atomic force microscopy, or AFM. There is nothing particularly atomic about this contrast mechanism in the nanoSQUID microscope- we don’t have nearly that much resolution- but it is incredibly useful for navigation because it allows us to safely and reliably detect surfaces without destroying the SQUID. Researchers and companies building scanning tunneling microscopes will often accomplish this by gluing their sensor, which is a microscopic metallic wire, onto a piezoelectric tuning fork and then exciting the tuning fork at its resonant frequency. This is a good strategy, but it must be modified for use with the nanoSQUID sensor, because the nanoSQUID sensor is considerably more massive than scanning tunneling microscope wires, so it cannot be glued onto the tuning fork without destroying its quality factor. We preserve the tuning fork’s quality factor by instead pressing a piezoelectric tuning fork against the side of the nanoSQUID sensor and performing shear force microscopy instead of tapping mode microscopy. The glass micropipettes serving as substrates for the nanoSQUID sensors are so thin that they bend easily when pressed agains the tuning fork, and this keeps them in mechanical contact with the fork. An optical microscope image of a nanoSQUID sensor pressed against a tuning fork is shown in Fig. 8.8A, and the resonant frequency of the piezo electrically driven tuning fork is shown in Fig. 8.8B, with a fit to a Butter worth Van-Dyke model. A phase-locked loop and PID feedback system together allow us to approach the surface with the nanoSQUID sensor, detect it without crashing into it and destroying the tip, and maintain feedback while scanning. Schematics of this assembly are shown in Fig. 8.9. A calibration of the scan range and height of the nanoSQUID AFM is shown in Fig. 8.10, with a comparison to a Bruker Icon AFM displayed as well.By far the most common experimental campaign for the nanoSQUID microscope during my time in Andrea’s lab involved being handed a sample fabricated primarily for transport or capacitance measurements, with little consideration afforded to the viability or ease of a scanning probe microscopy campaign on the sample. I think this is fairly common in scanning probe microscopy, and it often means that we need to get sensors to samples without much in the way of navigation infrastructure. For this reason the vast majority of nanoSQUID microscopy campaigns start with thermal navigation. Before cooling down the nanoSQUID microscope, drainage planter pot an attempt is made to align the nanoSQUID sensor with the heterostructure under an optical microscope, but the nanoSQUID sensor often still starts several hundred microns away from the sample. Once the system is cold, we generally proceed by injecting a few mBar of helium gas into the sample chamber.

This facilitates thermal transport between the nanoSQUID sensor and the sample. We then run an AC current through the sample, heating it and generating an AC temperature distribution. The nanoSQUID sensors are excellent thermometers as well as magnetometers, so we can use this thermal gradient to navigate to the sample. An image of the resulting distribution of temperature over the device is shown in Fig. 8.13A. Some of the details are described in a later section, but in summary this technique works surprisingly well- we can usually find samples even several millimeters away from the nanoSQUID sensor using this technique. Once the nanoSQUID is reasonably close to the sample, it is usually necessary to pump out the heat exchange gas before attempting magnetic imaging, since thermal contrast can produce large backgrounds. After the heat exchange gas is removed, further navigation must proceed by imaging the magnetic fields produced by applied current through the Biot-Savart effect, as illustrated in Fig. 8.13B.Thermal navigation does not work for all systems. In the simplest case in which other techniques are necessary, current cannot be driven through magnetic insulators, so if you want to find them with the nanoSQUID you must arrange for some navigation technique other than flowing current through the sample. There are a variety of solutions to this problem, and perhaps the simplest is fabricating an additional device adjacent to the one you’d like to investigate and running current through that instead. There are reasons you might want to avoid this- some samples are so unstable in air and moisture that it makes sense to avoid photolithography on heterostructures entirely- and for these situations, I’m going to discuss ferromagnetic navigation.There are a few engineering challenges associated with fabricating nanoSQUID sensors. I will briefly describe a particularly challenging one in this section. Many of the best elemental superconductors are soft, heavy metals with low melting points like lead and indium. As any person who has spent some time in an experimental physics laboratory knows, solder doesn’t wet too many materials well, and it certainly doesn’t wet glass, so these metals tend to form droplets when deposited onto glass substrates. To form a uniform film, the superconducting metal must freeze instantly upon landing on the glass micropipette. To make sure this occurs, we must cryogencially cool the glass micropipettes while evaporating the superconducting metal onto them. This process involves specialized machinery that is covered in great depth in other documents and publications, so I won’t discuss it here. However, I do want to discuss the nature of the failure modes of this process. When liquids don’t wet surfaces well, they dewet into droplets, and these droplets tend to get more spherical and less film-like the worse they wet the surface. If this process is allowed to proceed to its conclusion before deposited metal solidifies, the resulting films won’t be connected at all, and your nanoSQUID circuit will be open. If the substrate is cold enough, the resulting film will at least be continuous, and it is likely that you will get a nanoSQUID. However, the formation of droplets is impossible to completely stop, especially near the edges of films and on the oblique surfaces of the nanoSQUID sensor . These droplets generally won’t short the sensor, but the nanoSQUID sensor is so small that electrons can reach these droplets through tunneling processes. Whenever droplets form between the two superconducting contacts on the nanoSQUID sensor electrons can tunnel between the contacts through the droplet, with the droplet functioning as a quantum dot. The resulting Coulomb blockade phenomenon gives nanoSQUID sensors a very slight electric field sensitivity. Gating exfoliated heterostructures tends to produce large electric fields, and these are detectable as variations in the current through the nanoSQUID as a result of Coulomb blockade in parallel with the SQUID on the tip. Droplets functioning as quantum dots and producing a parasitic Coulomb blockage are so common that we observe them on nearly every nanoSQUID sensor, and we almost always have finite electric field sensitivity . This can be useful for finding the edges of devices in the absence of magnetism, but it is important to remember that not all nanoSQUID signals can be understood as local magnetic fields. Other parasitic contrast mechanisms do exist, but they are rarely dominant over magnetic or electric field sensitivity.

A subsequent 2017 multicenter RCT of 100 patients in the same groups also found similar pancreatitis rates but a 29.3% higher cost in the pancreatic duct stenting group, suggesting that early precut may be more cost-effective. The relationship between endoscopist experience and the success rates of pre-cut techniques were explored in six studies. Three found a positive correlation between cannulation success rates and endoscopist experience while the other three did not . In the meta-analysis by Sundaralingam et al, it was noted that only studies that involved expert endoscopists and not trainees showed a significant reduction in the risk of pancreatitis in the precut group compared with standard technique. Taken together, these studies suggest that in the hands of experienced endoscopists, early pre-cut techniques may facilitate SBC with lower PEP rates when compared to repeated attempts at standard cannulation. The TPS technique warrants additional discussion as this technique was not specifically evaluated in the studies included in the meta-analysis mentioned above. Through 5 studies, biliary cannulation success rates using TPS ranged from 85% to 100% and adverse events ranging from 3.5% to 20.5% and PEP rates ranging from 3.5% to 22.4% of cases. More recently, a meta-analysis comparing TPS to needle knife PP found significantly a lower success rate and a higher rate of bleeding complication for the needle-knife group but no difference in PEP, perforation, or total complication rates when compared to TPS. The authors conclude that TPS may have a higher SBC success rate with less bleeding but does not reduce the risk of PEP.Another type of rendezvous technique is the percutaneous rendezvous technique , pots with drainage holes in which access to the bile duct is achieved percutaneously , after which a guide wire is threaded anterograde through the needle into the bile duct and out through papilla.

This technique has been used in cases of difficult anatomy, e.g., patients with large, infiltrative tumors involving the papilla or cases of post-operative anatomy, such as Roux-en-Y anastomosis, Billroth II gastrectomy, where the location of the papilla may be difficult to access conventionally or via EUS. Case reports combining the Perc-RV technique with the placement of a hydrophilic guiding angiocatheter at the papilla to facilitate entry of the ST and/or percutaneous balloon dilation of the papilla via the anterograde direction prior to SBC have also been described. A retrospective analysis that looked at 24 patients with SBC failure due to many reasons found a 96% success rate with the perc-RV technique with a 2.4% complication rate . Perc-RV involves increased technical difficulty and training required to obtain percutaneous access to the biliaryducts, however, it allows a percutaneous transhepatic biliary drain to be inserted to alleviate biliary obstruction if other methods of obtaining biliary access fails . There are other methods of performing rendezvous-based SBC such as enteroscopy-assisted EUS or Perc-RV techniques for Roux-en-Y or Billroth II anatomy or intraoperative rendezvous technique where a guide wire is surgically inserted into the biliary ducts. These techniques are beyond the scope of this review, are generally reserved for advanced cases, and are typically performed in specialized endoscopy centers by endoscopists who have had extensive training and experience with difficult cannulation.ERCP has rapidly grown in its therapeutic abilities since its advent 50 years ago. One aspect that has stayed constant that both the first endoscopist to successfully cannulate the papilla, and most endoscopists today, would likely both agree is that successful ERCP hinges on adequate training, careful preparation in selecting the right patient for procedure, ensuring proper positioning and deciding initial techniques. Even with the ideal patient, up to 20% of SBC still fail when using conventional methods of contrast-assisted or WGC and are labelled difficult cannulation.

Although definitions vary, cannulation attempts of a duration over 5-10 min, over 5 attempts, or more than one unintentional MPD cannulation or opacification are the most widely used definitions for difficult cannulation. In this review, we have described steps to be considered and employed when difficult SBC is encountered. If the MPD has been cannulated, then a PD stent or CBD cannulation can be considered to reduce the risk of PEP. When the anatomy of the papilla is unfavorable or a difficult cannulation is anticipated, endoscopists can choose to perform a variety of early precut or rendezvous techniques, the choice of which depends on the experience of the endoscopist, disease being treated, and anatomy of the patient. The decision of which technique to pursue when difficult cannulation is encountered should include consideration of endoscopist experience, patient anatomy, and the disorder being treated, among other factors.Bunch grapes , notably European , are considered among the major fruit crops worldwide, producing roughly 70–80 million tons each year . Cultivars of V. vinifera L. are used for wine, juice, and table grape production. Grape berries are classified as nonclimacteric fruits, exhibiting a double-sigmoid developmental pattern with two rapid growth phases: the berry formation and the ripening phase , separated by an intermediate lag phase called the green plateau . The exponential increase in berry size characterizes both growth stages , but not the lag one . During phases and , also known as immature stages, organic acids, mainly tartrate and malate, accumulate leading to induction of acidity levels . At the end of the lag phase, a step-change point takes place known as veraison, where acidity starts to decline while sugars, mostly glucose and fructose, as well as anthocyanins in colored varieties, increase. Of particular interest are phenolic compounds, which are major and ubiquitous plant secondary metabolites derived from the shikimate/phenylpropanoid and polyketide pathways, with three utmost categories: proanthocyanidins , also known as condensed tannins, the gallo- and ellagitannins , and the phlorotannins . Such diversity of polyphenols, with more than 8000 structural variants, bestows them a wide range of biological functions ranging from growth, development, and protection inside the plant to, to some extent, human-related issues . In grapevines, the accumulation pattern of phenolic compounds, along with the aforementioned berry attributes, distinguishes each of the berry phases throughout berry development . Indeed, berry quality and sensory characteristics are notably defined by its polyphenol content . Remarkably, astringency is among the hardest sensory traits to depict and interpret as many intricate processes underpinning its perception . For instance, a sensory characterisation of the astringency of 11 varietals of Italian red wine revealed that neither total phenols nor PAs can predict how all astringency subtleties will be perceived . It is worth noting that the amounts, compositions, and proportions of polyphenols in a given species may vary widely depending on several factors, such as genotypic variations, developmental stages, and environmental circumstances . Scarlet Royal is a mid-season ripening table grape variety, producing seedless, red-skinned, oval-shaped, firm, and moderate to large berries with a sweet to neutral flavor .

In the San Joaquin Valley, California, it typically ripens in mid to late August, filling the harvest window between Flame Seedless and Crimson Seedless, and has thus become a very popular red table grape variety in California. However, an undesirable astringent taste has been observed occasionally in some cases. In fact, the economic value of grapevines depends substantially on the environmental conditions, including climate, soil, cultural practices, cultivar, and rootstock. Hence, the term “terroir” is used in viticulture to describe the effect of such an interactive ecosystem on grapevine and wine quality . The current study aimed to understand the underlying mechanism of astringency development in Scarlet Royal berries at two contrasting vineyards . The first location produces well-colored, non-astringent berries; however, drainage pot the second site yields astringent taste, poorly colored berries . The data showed a large variation in berry astringency within the same vineyard and from year to year. The data illustrated that the divergence in berry astringency stemmed from alterations in its polyphenol composition , most notably tannins. Additionally, the ripening stage was the most distinguishing platform for such variation between both vineyards. We were able to determine the tannins’ threshold level that causes the Scarlet Royal astringency taste to be ~ 400 mg/L. Given the changes in the levels of polyphenols during berry ripening, the question was raised: what is the mechanism governing the distinctive tannins accumulation pattern between V7-berries and V9-berries, and hence astringency diversity? To answer this question, RNA-seq data generated at one ripening time point was associated to the changes in polyphenolic levels using a systems biology approach, WGCNA . The module-trait association analysis positively correlated the key flavonoid/PAs biosynthetic genes with the accumulation of tannins, catechin, and quercetin glycosides exclusively in V9-berries. The modulation of the berry’s transcriptomic profile is concomitant with its polyphenols’ composition, which finally disturbs berry quality, including astringency levels.Five-year-old V. vinifera cv. Scarlet Royal grafted on Freedom rootstock was chosen for its berry astringency diversity at two commercial vineyards located in Delano, San Joaquin Valley, California, USA. Vineyards were located at a close distance of 10 km, and the local weather conditions during the two seasons were collected from the Delano CIMIS weather station . Both vineyards were planted at the spacing of 2.44 and 3.66 m in an open gable trellis supporting system with East-West row orientation. Vines were pruned in a Quadrilateral cordon training with 7–8 spurs left on each cordon during the winter pruning. In addition, general UC guidelines practices were applied in both vineyard. Starting from veraison and until the end of the season, during two consecutive years . During the first year, sampling dates were July 8th , August 1st , August 10th , September 9th , September 15th , and October 19th ; and for the second year, sampling dates were: July 15th , August 10th , August 25th , September 10th , September 29th , and October 21st . Sampling dates varied from the first to the second year due to the vineyard’s accessibility. At each sampling point, two sets of fifty berries were collected periodically. The first set was used to measure the berry weight, and then these berries were macerated in an electric blender, filtered through a paper towel, and an aliquot of juice was used to determine soluble solids , pH, and titratable acidity . Soluble solids were determined using a tabletop Milwaukee MA871-BOX digital refractometer . The TA and pH were determined by titrating a 40 mL aliquot of juice with 0.1 N NaOH to a pH of 8.2 using an automatic titrator Excellence T5 . Another random 50 berries from each replicate were collected for color, tannins, and phenolic compounds and sent immediately in a cooler to EST laboratories. At harvest, which was during the month of September, an extra set of samples was collected and promptly frozen in liquid nitrogen and stored at −80°C for subsequent analysis, including RNA extraction and gene expression studies. Harvest time was determined by the growers, and the marketable clusters were picked based on the color, and yield was determined from the three harvest dates.At bloom, fifty leaves from each replicate were collected, resulting in a total of 200 leaves from each vineyard, for nutrient analysis. The leaf positioned at the front of the cluster was specifically selected, and the petiole was immediately separated from the blade. The petioles were transported to the laboratory, where they were triple-washed with distilled water to remove any impurities before being sent to a private laboratory for nutrient analysis. In the winter, soil samples were collected at a depth of 30 cm and at a distance of 30 cm from the vine. These samples were transported immediately to the laboratory for analysis. The nutrient content was determined using the methods described in US Salinity Laboratory Staff .The taste panel evaluation of Scarlet Royal table grapes was conducted with the participation of twelve nonprofessional panelists. Astringent taste perception was assessed using a scale ranging from one, representing an extremely low level of astringency, to seven, indicating an extremely high level of astringency. The taste evaluation was performed on 24 clusters from each vineyard. Phenolic compounds analysis. Total phenolic analysis was performed on 250 grams of whole berries by ETS laboratory using a reversed-phase HPLC method adapted from Price et al. .Total RNA was isolated from whole berry samples following the protocol described by Boss et al. . To remove any residual DNA, RNase-free RQI treatment was performed according to the manufacturer’s instructions , and the samples were further purified using the RNeasy mini kit .

Elucidating the process by which family members managed growing incompetence sheds light on the important role of awareness perception in household dementia care. Currently, there exists an extensive literature examining clinical dimensions of declining deficit awareness or anosognosia, including its detection and measurement, especially for diagnostic purposes , cognitive correlates and the nature of its expression, including its fluctuations and effects on self . Despite these research gains, there are currently few studies that address the impact that perceived anosognosia has on family care giving and none that elucidate the social process by which it occurs. Related work, for instance, shows that anosognosia is associated with increased caregiver burden . The current study extends this area of research by showing the central role that lay perceptions of awareness play in efforts to manage growing incompetence in everyday activities. It explains the social mechanism by which the perception of declining awareness transforms care from a collaborative pursuit with the declining individual into a unilateral pursuit utilizing an array of tricks and ruses to influence the individual’s activity choices. The study’s naturalistic design helps show the importance of the interpretive process in assessing the household impact of a clinically-defined symptom, suggesting one reason why the clinical presence of anosognosia would not always translate into caregiver burden. This study’s insight about the interpretive challenges that families encounter around deficit awareness holds important epistemological implications for a common clinical approach to identifying and measuring anosognosia. The approach measures deficit awareness using discrepancy scores generated by comparing the elder’s self-rated scores on daily functional abilities with the scores rated by a caregiver . In this approach, vertical gardening in greenhouse the caregiver’s rating is treated as an accurate assessment and its discrepancy with the elder’s score reveals to what extent he or she is not aware of his or her own ability decline.

The current study however, suggests that there can be problems with treating the caregiver’s report as fully reliable. The findings show that caregivers may sometimes have difficulty grasping an individual’s level of decline. First, family members themselves may suffer from a kind of courtesy anosognosia, a state in which they are unwilling or unable to admit to the extent of a loved one’s decline. Multiple family members in this study, for instance, reported that the individual’s decline was denied by others in the household when met with arguments or plausible evidence to the contrary. Second, family members also signaled interpretive troubles by describing moments when elders employed a likely cover-up tactic to hide small errors in ability. They attempted to hide errors by taking issue with or showing nonchalance around what others defined as a “correct” procedure for accomplishing a task. In this way, affected individuals attempted to resist or redefine normative measures of successful activity engagement. One family member, for instance, suspected this maneuvering when her mom served her dad cold soup and claimed that he liked it that way. Another participant reported her dad’s nonchalance or apparent lack of care after she pointed out that he did not have to have coffee with the grounds in the coffee cup.Facing a disease that is deeply stigmatized, individuals with Alzheimer’s have social and psychological reasons as to why they would hide their awareness of errors in ability from others . Efforts taken to avoid acknowledging the disease’s effects, for instance, may signal the elder’s effort to protect him or herself from social and/or psychological injury . While these interpretive obstacles pose challenges to using caregiver reports of ability level, this study suggests that to better understand the interpretive challenges that observers face and thus the reliability of observer reports, researchers must better understand the social process in which the interpretations are made. These findings call for more research around the interpretive challenges that proxy raters may encounter. While researchers have highlighted the issue of duplicity in care giving and the kinds of ethical dilemmas it can create , the current study identifies the turning point in the social process of care when family members began using duplicitous techniques.

The evidence shows that they began using these techniques as a self-conscious strategy when the elder’s awareness ability was thought to have declined severely enough to make transparent negotiation difficult. At this point in the progression of declining deficit awareness, family members found that elders were less capable of heeding their warnings and following their instructions for avoiding activities that had become too dangerous to pursue independently. At this stage, family members resorted to techniques that function as ruses to fool or trick elders away from risky activities. This study has two important limitations. First, the participant sample is almost entirely made up of adult-children. It is likely that adult-children will experience a more protracted and potentially more risky transition period between collaborative and unilateral autonomy management work in contrast to spousal caregivers. Adult-children commonly must make decisions in concert with other siblings or family members. Negotiating with multiple members may raise the likelihood of disagreement and conflict that can delay a decision to curtail an elder’s independence when it is urgently needed. In addition, spouse caregivers likely will have spent many more years in close proximity with the affected individual and be better suited to notice minute, yet critical changes in the individual’s awareness level that can lead to timely changes in their autonomy management practices. Second, the participant sample only includes family members dealing with individuals who have one type of dementia: Alzheimer’s. While there are important differences in symptom behaviors between dementia sub-types , we believe that these results would extend to families dealing with vascular, Lewy body, and frontotemporal dementias. Future research can test the extent to which features of the autonomy management process identified within the Alzheimer’s context can effectively explain features of the support process around other forms of dementia. This study reveals the social contextual factors that influence how family members identify and respond to growing signs of risk in an elder’s decades-old activities. Across theautonomy management process, family members attempted to balance their concerns of reducing risk with their concerns of permitting enough autonomy to promote wellbeing. In limiting an elder’s activities too much, for instance, they feared they could reduce his or her chances of finding spontaneous sources of meaningful engagement and potentially cause friction within their relationship. Yet in limiting their activities too conservatively, family members feared they could inadvertently permit elders to injure themselves and possibly others. Autonomy management provides a useful conceptual framework for understanding how family members adjusted their measures to deal with this dynamic as the disease advanced.The fresh market berry industry in Santa Cruz and Monterey counties is an excellent example of transformation in the business of agriculture over the last 50 years. Located along the Central Coast of California, the two counties span the fertile Pajaro and Salinas valleys, and are well known for their amenable climate and production conditions, their diverse crop mix and grower demographics, and their developed agricultural infrastructure and support industries. The majority of the berry sector is comprised of strawberries , raspberries and blackberries , with blueberries and other miscellaneous berries produced on a much more limited basis. Substantial research-based literature and historical information is available for Central Coast strawberries; however, despite the area’s move towards greater production of raspberries and blackberries, less information exists for these crops. We seek here to provide a more complete portrayal and historical context for the berry industry in the Santa Cruz and Monterey area, greenhouse vertical farming which is the origin of the berry industry in California. While the berry industry has been very successful in recent decades, it now faces new challenges, such as invasive pests and the phaseout of the soil fumigant methyl bromide. This article draws on previous and more recent research to discuss some of the influences that have contributed to the berry industry’s dramatic expansion in Santa Cruz and Monterey counties, including selected innovations in agricultural practices and heightened consumer demand.During the 1960s and 1970s, the number of acres planted to berries, tons produced and value of production fluctuated.

The fluctuations can be partly explained by farm management: in the past growers often rotated berry and vegetable crops to assist with soil and pest management, thereby influencing these statistics. However, annual crop reports from the county agricultural commissioners show that since the 1980s, berries have become increasingly important to each county’s overall value of production, and by 2014 accounted for 64% and 17% of the total value of all agricultural products in Santa Cruz and Monterey counties, respectively . The industry’s growth can be explained by a shift of some acreage out of tree fruits and field crops , among others, into berries, and by additional acreage put into agricultural production.Strawberries are the undisputed leader in the berry sector and in 2014 represented 58% and 94% of the value of all berry production in Santa Cruz and Monterey counties, respectively , and 50% and 93% of all berry acreage . Table 2 documents the remarkable expansion of the strawberry industry over time in both counties with respect to acreage, tons produced and value of production. Between 1960 and 2014, acreage more than tripled and production increased tenfold. The value of production, in real dollars, increased by 424% in Monterey County and by 593% in Santa Cruz County, reaching an astonishing combined value of nearly $1 billion in both 2010 and 2014. The gains in all statistical categories in Monterey County were enabled in part by an expansion of production into the southern reaches of the county where more and larger blocks of farmland are available, and where land rents are lower than in Santa Cruz and northern Monterey counties. However, from 2010 to 2014 Monterey County’s tonnage and production values declined, possibly because the area has recently experienced a shortage of labor to harvest fresh market crops. Tonnage was also lower in Santa Cruz County, but production values increased. This may be because of the county’s greater emphasis on local agriculture, organic production and direct market sales, which are often associated with higher crop values. For raspberries, the acreage, tons produced and value of production grew steadily and most strikingly in Santa Cruz County , where production conditions for caneberries are optimal. For example, caneberry fields in Santa Cruz County are situated in areas that have well-drained soils and are protected from damaging winds. Also, fields are planted to take advantage of the growth and yield gains associated with southern exposures. Moreover, field-to-cooler travel distances are shorter in Santa Cruz County, which is critical for safeguarding the quality and marketability of these highly perishable crops. By 2014, raspberries represented 33% of the county’s total value of production for all berries. In contrast, Monterey County raspberry production accounted for only 6% of the county’s total berry value. Blackberries have not been consistently reported as a separate category in archived statistical analyses, but instead were often included under the terms “bush- or miscellaneous berries”. Therefore, similar data for blackberry acreage and value of production cannot be reported here. However, between 1990 and 2010, Santa Cruz County agricultural commissioner crop reports reported an upward trend for the broad category with respect to acreage planted and value of production . In 2010, blackberries were promoted to a position of prominence in the report and shown as a separate statistic; at the same time, the miscellaneous berry category was shown to be very small indeed. Between 2010 and 2014, however, blackberry acreage and value of production leveled off and have shown only modest gains . This may be because there has been less emphasis on production and market research and promotion for blackberries than for strawberries or raspberries. No comparable data are available for Monterey County. The two counties have contributed significantly to California’s total berry sector: in 2014, area strawberry acreage represented 35% of the statewide total, 37% of the total tons produced and 38% of the total value of production . Area raspberry acreage represented 43% of the statewide total, 42% of the total tons produced and 39% of the total value of production. Comparable statewide statistics are not available for blackberries. County agricultural commissioners’ reports show that the majority of all berries produced in the two counties — up to 98% — are sold as fresh market fruit .

Whenever possible, individuals located more than 2m from trails and fuel breaks were selected to avoid any edge effects. All individuals were sampled once between April and September 2019. Two branchlets , each containing necrotic lesions and adjacent asymptomatic wood tissue, were clipped per individual using sterile techniques, for a total of 600 samples. Samples were retrieved from approximately breast height and opposite sides of the shrub, whenever possible. All individuals had at least two necrotic lesions, even if no significant dieback was observed, allowing these methods to be carried out across all 300 individuals. Samples were then placed in labeled plastic bags, stored on ice, and brought back to the lab and placed in a 4o C refrigerator.Samples were rinsed of dirt and debris and surfaced sterilized using 100% ethyl alcohol, 0.5% bleach, and a 70% ethyl alcohol rinse. Cross sections between 1-2 mm were isolated from the advancing canker margin and plated onto half-strength potato dextrose agar amended with streptomycin antibiotic. Cultures were incubated at room temperature until fungal colonies developed , and isolates of hyphal tips near the advancing margin were then re-plated onto half-strength PDA-strep to obtain pure cultures. From pure culture, any samples identified to have morphological characteristics consistent with those of Bot. fungi  were selected for PCR. A few isolates from cultures inconsistent with Bot. characteristics were randomly selected from each site to amplify and sequence to verify our morphotyping method. The internal transcribed spacer region 1 and alpha-elongation factor-1 genes were amplified using PCR primer pairs ITS1F and ITS4, 25 liter pot and EF1-728F and EF1-986R, respectively, using methods modified from White et al., and Slippers et al .

Successfully amplified samples were sequenced using the UC Berkeley Sequencing Facility .The severity of Bot. infection was calculated as the isolation frequency per site. Data were square-root transformed when necessary to meet the assumptions of normality. Differences in mean Bot. infection severity between elevation categories were calculated using one-way ANOVA with Tukey’s HSD for post-hoc analysis with R Statistical Software . Correlations between actual elevation and Bot. infection severity were assessed using simple linear regression and ANOVA to test for significance . Generalized linear models were developed to identify patterns of dieback, with dieback severity values as the response variable, and elevation , Bot. infection severity , and aspect as possible explanatory variables. If multiple models received substantial support , the best model was confirmed by calculating the relative importance of each term based on the sum of their Akaike weights . The proportion of variance explained by the models was calculated by measuring the adjusted D2 value .This study provides definitive support for the hypothesis that shrub dieback, during a recent drought, and pathogen infection are strongly related in a wild shrubland setting. This is the first known quantitative support for the hypothesis that in A. glauca, an ecologically important shrub species in the study region, dieback is related to pathogen infection occurring along an elevational gradient. As expected, N. australe and B. dothidea were the two most frequently retrieved pathogens across all sites, however N. australe, the introduced pathogen, had almost twice the abundance of B. dothidea. N. australe is driving the correlation between elevation and Bot. infection, as the frequency was greater at lower elevations compared to upper elevations, while B. dothidea abundance did not change significantly across elevations. Level of Bot. infection was confirmed to be a significant predictor of stand-level dieback severity. The data also confirm that stand dieback severity is generally greater at lower elevations, which in this region experience higher temperatures and lower annual rainfall than the higher elevations sampled.

While the presence of Bot. species has been reported previously in Santa Barbara County, this study represents the first effort to understand the abundance and distribution of Bots occurring in natural shrublands, and the first wildland shrub survey of Bots across a climate gradient. The high frequency and wide distribution of Bots retrieved from our study sites support the hypothesis that Bot. species are widespread across a natural landscape, and likely contributing to the extensive dieback resulting from the recent drought. Bot. fungi were retrieved from nearly every site in this study . We could not determine Bots. presence from three sites due to contamination issues. The broad extent of the study area suggests that infection is widespread in the region, and likely extends beyond the range of our study. While both N. australe and B. dothidea together made up the most frequently retrieved pathogens, our data show that N. australe has a larger distribution and occurs in greater abundance across the study region than B. dothidea . This trend was consistent across all elevations, but particularly at lower elevations . One possible explanation for this is that N. australe, being a recently introduced pathogen, spreads more rapidly as an exotic species in A. glauca compared to B. dothidea, which has been established in California for over 150 years . This hypothesis is consistent with previous studies that have shown variations in Bot. species abundance and virulence in Myrtaceous hosts occurring in native versus introduced ranges . However, it is difficult to evaluate the incidence of B. dothidea and N. australe in the present study in relation to historical documentation since many species in the Bot. complex have, until recently, been mischaracterized . Only with the recent development of molecular tools have researchers begun to accurately trace phylogenetic and geographic origins of Bot. species. Such studies are beginning to elucidate the complex existence of Bot. fungi as both endophytes and pathogens around the world, and much more research is needed to understand their pathogenicity in various hosts under different conditions. Nevertheless, it remains clear from our study that Bot. species, particularly N. australe, are both abundant and widely distributed in this region, and are important pathogens in A. glauca shrubs.Because Bot. taxa were the most frequently retrieved pathogens and were significantly correlated with dieback, we believe that they drive A. glauca dieback. Further, stand dieback severity increased significantly with Bot. infection. This is not to say that other pathogens do not also contribute to disease symptoms, but we found no evidence of any other pathogens occurring in such high incidence as Bot. species. While Brooks and Ferrin identified B. dothidea as a likely contributor to disease and dieback in dozens of native chaparral species during an earlier drought event in southern California, and Swiecki and Bernhardt found B. dothidea in association with a dieback event in stands of Arctostaphylos myrtifolia in northern California, our study yields the most extensive results of Bot. infection and related dieback in a chaparral shrub species across a landscape. Further, our study resolves species identity within the Bot. clade and highlights the role of the recently introduced pathogen, N. australe.A significant finding in this study was the relationship of Bot. infection and dieback with elevation. Bot. abundance and dieback were both found to be greatest at lower elevations, raspberry cultivation pot which was driven mostly by the high frequency of N. australe retrieved at these sites. This represents the first quantitative evidence supporting that A. glauca vulnerability to fungal infection is influenced by stress levels along an elevation gradient.

A similar pattern was observed in northern California by Swiecki and Bernhardt , who suggested that dieback in Ione manzanita infected with B. dothidea was greater in drier sites compared to more mesic ones, although no comparison of infection rates between sites was conducted in their study. The elevation gradient in our study was used a proxy for stress levels because annual precipitation decreases with decreasing elevation within our study region . Higher temperatures, which are associated with lower elevations, are also known to play an important role in drought-related mortality, as water loss from evapotranspiration is increased . Furthermore, unpublished data for dry season predawn xylem pressure potentials on a subset of sites along the same elevational gradient revealed more negative water potentials in A. glauca at lower elevations compared to upper elevations as spring and summer drought sets in . Thus, there is evidence that shrubs at low elevations indeed experienced the greatest water stress during the 2011-2018 drought, which predisposed them to higher levels of Bot. infection and enhanced dieback compared to upper elevation sites. More in-depth studies on the microbial communities and fungal loads of healthy and diseased shrubs throughout the region would help elucidate such trends. Another possibility for the higher incidence of Bot. infection at lower elevations is that the lower ranges of A. glauca populations in Santa Barbara are often located adjacent orin close proximity to agricultural orchards, ranches, and urban settings, which are common sources of plant pathogens, including Bots . Eucalyptus, avocado, and grapevines, which are abundant in these areas, are particularly well-known Bot. hosts and potential facilitators of Bot. introduction . Therefore, sources of inoculum from nearby populations of agricultural and horticultural hosts could be responsible for continual transmission Bots in wildland A. glauca populations, and would likely result in greater rates of infection at lower elevations. Furthermore, many of the lower sites in the survey were located near roads and/or trails, which are often subjected to additional stress from human activity like pruning and trail clearing; activities that are known to spread and promote infection by Bot. pathogens . While we avoided sites that showed signs of such activities in our survey, we cannot rule out the potential contributions of proximity to human encroachment to the overall higher rates of Bot. infection across the lower elevation zone. It is worth noting that while our study revealed a trend of increased dieback in lower elevations, some upper elevation sites also exhibited high levels of dieback, and Bot. fungi were retrieved from many of these sites. Upper elevations also experienced significant stress during the 2011-2018 drought, and water-related microsite variables outside the scope of this study like slope, solar incidence, soil composition, and summer fog patterns factors likely contributed to increased stress and subsequent dieback. Additionally, N. luteum, N. parvum, and D. sarmentorum were isolated primarily from upper sites. Host plants in these sites may serve as potential reservoirs for disease because the milder climate conditions promote greater host survival and thus pathogen persistence asendophytes. This serves as an important reminder that continued global change-type drought may eventually jeopardize susceptible species populations even at the upper boundary of their range.Our results are consistent with well-known theoretical models describing the relationship between environmental stress and biotic infection, which generally ascribe extreme drought stress as a mechanism for plant predisposition to disease . These frameworks illustrate dynamic interactions between environmental stress, plant hydraulic functioning and carbon balance, and biotic attack, and a growing body of research has focused on understanding the roles of these factors in driving plant mortality, especially during extreme drought . While the data collected in this study do not directly address the specific mechanisms leading to Bot. infection and dieback in A. glauca, our results can be discussed in the context of how life histories and physiological adaptions elicit differential responses to drought in woody plants, particularly in chaparral shrubs. For example, shallow-rooted, obligate seeder shrubs like A. glauca have been shown to be more susceptible to drought-induced mortality during acute, high intensity drought than deep-rooted, resprouter shrubs . This supports our observations of pronounced A. glauca decline during an historic California drought compared to nearby resprouter species like chamise , and laurel sumac .Additionally, physiological mechanisms related to drought tolerance may further explain predisposition to disease in A. glauca. For example, high resistance to cavitation is a common trait associated with more dehydration-tolerant species like A. glauca that maintain hydraulic conductivity during seasonal drought . While cavitation resistance is thought to assist in the continuation of photosynthetic activity even at very low seasonal water potentials , it has also been associated with greater mortality rates during high intensity drought in a variety of woody plant systems including mediterranean shrublands , temperate deciduous forests and eucalyptus forests . High resistance to cavitation requires heavy carbon investment for stronger and denser stem xylem tissue , which can result in limited carbon for investment in defense against pathogens like Bot. fungi.

Moore & Goodson followed children with ASD from two to four years old and found that overall rates of RRBs reduced, with low level RRBs significantly reducing, yet different RRB subtypes persisted in a more complex form. Whereas Honey, et al. examined preschoolers and found within a year there was a significant decrease in the severity of RRBs observed. However, a more recent study, which utilized observational coding of RRBs in a preschool aged sample, found there was no significant change in any of the RRB subtypes coded across 13 months and three assessment time points . Further, Richler, et al. used the ADI-R to track change in RRBs over a period of 9 years and found that low level sensory motor RRBs actually remained high. Taken together, these findings suggest that the developmental progression and transformation of RRBs overtime within individuals remain unclear, with the biggest influence being the sample population and measurement tools used .The general consensus within the field is that children with more severe adaptive and cognitive impairments exhibit higher frequency, more intense, and more persistent RRBs . More specifically, children with lower cognitive capacity exhibit the most frequent and severe low-level RSM RRBs, whereas children with higher cognitive capacity exhibit significantly less of the RSM behaviors . However, these findings are nuanced, as there is not a singular and linear relationship between IQ and RRBs, and consideration must be given to the types of RRBs being measured. For example, grow bucket in a study examining 830 children with ASD between 15 months and 12 years old with an average age of 5 years old, found that for many RRBs, a significant interaction effect was found between nonverbal IQ and age .

Specifically, in older children, NVIQ was strongly related to low-level RRBs such as hand and finger mannerisms. However, high-level RRBs like circumscribed interests were positively related to NVIQ. Bishop, Richler, & Lord used the ADI-R to examine the total as well as the individual types of RRBs, which included 13 types of behaviors considered to fall under the RRB umbrella. Another interesting finding from this study was the relationship between NVIQ and RRBs actually became stronger with increasing age, where children under the age of 3 showed no relationship between RRBs and NVIQ. Similarly, Kim and Lord found no association between NVIQ and RRBs in toddlers under two years old. Taken together, these findings further evidence the importance of measuring subtypes individually, as there are clear differences across RRB types in the relationship between cognitive ability and RRB presentation. Findings across studies highlight the importance of examining specific subtypes of RRBs and the traits associated with them, as these traits may significantly impact the persistence or the possible reduction of RRBs overtime. For example, Ray-Subramaian and Weismer found that not only were receptive and expressive language skills significantly lower among children with higher rates of RRBs, but they also concluded that higher scores in both language domainsin 2-3 year olds could significantly predict a reduced rate of RRBs. Again, consumers of this area of ASD research must take into account the number of participants and age span included across studies. Most recently, a study examined children at three time points to determine if RRB presentation at 1-2 years old, and/or 3-5 years old can predict cognitive functioning, adaptive skills and ASD symptomology at 8-10 years old . Results showed that increased severity of low-level RRBs were significant predictors of lower cognitive and adaptive skills as well as a greater ASD symptom severity at age 8-10 years. This relationship was not found when examining whether RRBs in the first two years of life could significantly predict the same school-aged outcomes .

Despite the nuanced findings among studies regarding the specific relationship and influence RRBs have on cognitive performance, it is clear that future studies must consider and control for cognitive functioning when examining the relationship between RRBs and other clinical characteristics .Children with ASD exhibit significant impairment in adaptive functioning skills that extend beyond their cognitive deficits . It is important to note that adaptive functioning skills measure the ability of an individual to successfully function within their given environment, and studies have demonstrated greater deficits in adaptive functioning for children with ASD compared to age and IQ matched peers . Several studies have examined the relationship between adaptive skills and RRB presentation in individuals with ASD . Similar to the relationship between RRBs and IQ, results have varied based on the measures used and age of participants; however, it can be deduced that in general, higher rates of RRBs are associated with lower adaptive functioning skills . However, this finding has varied across studies based on the age and IQ of the participant . The relationship between adaptive skills and RRB presentation in ASD is complex and has varied results based on the age and IQ of participants as well as the measures employed. For example, Liss, et al. found that the relationship between adaptive functioning and RRBs was dependent on the severity of adaptive skill impairment. Specifically, there was no significant relationship between RRBs and adaptive functioning in lower functioning children with ASD; yet the high functioning group exhibited a significant correlation between adaptive behaviors and RRBs. Few studies have examined adaptive functioning and RRBs using measures other than parent report. However, in young children, self-regulation has been observed in interactions between parent and child. As shown by Wetherby and Prizant , children with ASD exhibited lower proportions of well-regulated behavior bouts and higher incidences of RRBs during parent child interactions. Theoretically, some have suggested that specific RRBs may be a result of an emotional trigger for children with ASD. However, Militerni, et al. found that most of the low-level RRBs observed in 2-7 year olds were not reactive to a particular emotional trigger. The remaining 29% of RRBs deemed to be reactive in nature consisted of high intensity sensory behaviors, including self-injurious behaviors, motor RRBs and sensory stimulation, which were all more common the younger participants . This notion of an emotional trigger also highlights a theory that RRBs serve as a coping strategy to regulate their state of arousal; however, there are currently not enough results or data to full endorse this theory and further examination is needed .

There are a number of common developmental and neuropsychiatric disorders that overlap in symptom presentation, and in some cases are determined to co-occur in children with ASD. Some of the most common are attention deficit hyperactivity disorder and anxiety disorders . There is limited knowledge about how these ASD-related disorders vary across the population and what impact the co-occurring conditions impact RRB manifestation as well as the impact on overall development, adaptive skills and other child characteristics. Attention deficit hyperactivity disorder . Attention deficit hyperactivity disorder is a neurodevelopmental disorder characterized by symptoms of inattention, impulsivity, and/or hyperactivity exhibited to a degree substantially beyond what is expected for developmental level . ADHD and ASD share overlapping symptoms such as issues with communication problems, issues with attention and the presence of restricted behaviors . Although the last version of the Diagnostic and Statistical Manual of the American Psychiatric Association prohibited a dual diagnosis of ASD and ADHD, dutch bucket for tomatoes preliminary evidence suggests that when these two disorders co-occur, the risk for increased severity of psychosocial issues intensifies . Such findings are in conjunction with a growing number of researchers reporting children who meet criteria for both disorders are evidence to suggest they can co-occur .The need for research to examine the dual presence of clinically significant ADHD symptoms in individuals with ASD has begun to be addressed . This study compared school-aged children 4 to 8 years old that included younger siblings of children with ASD , children with ASD , low-risk controls and children with language delay to include reference points for functioning and skill level across groups. Results indicated that children with comorbid ASD and ADHD diagnoses had lower cognitive functioning, more severe social impairments, and greater delays in adaptive functioning than children with ASD only . There is a great need for continued exploration of the impact of co-occurring ASD and ADHD symptomology on children with ASD; specifically, how elevated levels of hyperactivity influence the presence and severity of RRBs in children with ASD. Anxiety. The role of anxiety for individuals with ASD has been proposed to play a key role in the severity of RRBs, as the function of engaging in specific RRBs has been hypothesized to serve as a coping mechanism to reduce feelings of anxiety . However, it should be noted that there is insufficient evidence currently to support this theory . Scientific evidence illustrating links between anxiety, ASD and RRBs is limited. However, there have been studies that indicate high levels of anxiety in the ASD population and even links to symptom severity increasing . The link between anxiety and ASD symptom severity is logical considering the need for routine, sameness and consistency to a severe degree. Interruption of those may result in increased levels of anxiety and intense stress often accompanied by outbursts when self-control is impaired. The theories accounting for the popular notion that anxiety and arousal states significantly contribute to increased RRB severity for individuals with ASD still needs to be explored .The present study aimed to explore the phenotypic presentation of RRBs and associated characteristics for individuals with ASD between 4 and 18 years old. Previous studies have been limited by measurement tools, limited age included, as well as limited statistical power due to smaller sample sizes; therefore, this study aimed to examine the forms of RRBs across age and IQ, and to examine the impact of hyperactivity, anxiety, coping skills, and ASD severity on RRB presentation in a large, well-characterized sample of individuals with ASD. The first aim was to define the specific RRB subtypes derived from a factor analysis of the Repetitive Behavior Scale- Revised . Secondly, the RRB subtypes derived from the factor analysis were used to cluster participants based on type and severity of co-occurring RRB subtypes into phenotypic profiles. The final aim of this study was to explore the role of clinical , cognitive and adaptive skills in predicting phenotypic profile group membership. Researchers have yet to uncover the specific function/s of RRBs; therefore, examination of the predictive power of individual clinical characteristics on RRB phenotypes contributes to this area of research.The first aim of this study was to examine the factor structure of the Repetitive Behavior Scale- Revised to determine how many unique RRB forms are measured. The first step in determining the factor structure was to run an exploratory factor analysis of the 43 RBS-R items using Mplus Version 7 , an oblique CF-quartimax rotation and a weighted least- squares with mean and variance adjustment to account for the ordinal nature of the data . EFA assumes that each variable, in this case each question on the RBS-R, may be associated with any other factor without an a priori hypothesis about factors or variables . To determine the optimal number of factors, a combination of model fit statistics and examination of factor loadings were used. The chi-square value is typically an informative model fit statistic; however, the chi-square test is sensitive to sample size, such that large samples often result in statistically significant chi-square values . Given the number of participants in the current dataset, the chi-square values were analyzed with caution. Additional model fit statistics included root mean square error of approximation , the Standardized Root Mean Square Residual , the Comparative Fit Index and the Tucker Lewis Index . The RMSEA is a measure of model fit that is not as sensitive to sample size and values below .06 indicate an acceptable model fit . The SRMR is another descriptive model fit statistic in which lower values indicate better model fit, with a suggested cut-off of .08 or below . Lastly, both the CFI and TLI are typically presented together in EFAs and both serve as measures of model fit, ranging from 0 to 1 with higher values indicating better fit and cutoff scores of .90 .Determining Factor Structure. As items were permitted to load on only one factor for the CFA, items that loaded significantly >.30 on more than one factor were evaluated to determine the ideal factor pattern.

CBF1 transcript abundance is very sensitive to chilling temperatures; it is a master regulator of the cold regulon and improves plant cold tolerance . PIF7 binds to the promoter of CBF1, inhibiting CBF1 transcript abundance, linking phytochrome, the circadian clock and CBF1 expression. Our data are consistent with this model; transcript abundance of PIF7 was higher and CBF1 transcript abundance was lower in BOD berry skins than RNO berry skins .ABA concentrations in plants increase in response to dehydration and ABA triggers a major signaling pathway involved in osmotic stress responses and seed development. ABA concentrations only increase in the seed embryo near the end of seed development when the embryo dehydrates and goes into dormancy. ABA concentrations remain high to inhibit seed germination. The transcript abundance of ABA signaling genes such as ABF2 and SnRK2 kinases increase after application of ABA to cell culture and in response to dehydration in leaves of Cabernet Sauvignon. The data in this study are consistent with the hypothesis that BOD berries are riper at lower sugar levels. The ABA signaling genes in the berry skins had higher transcript abundance in BOD berries indicating that ABA concentrations were higher in BOD than RNO berries even though RNO berries were exposed to drier conditions . ABA concentrations may be higher in the BOD berry skins based upon the higher transcript abundance of important ABA signaling and biosynthesis genes encoding ABF2, SnRK2 kinases and NCED6. We hypothesize that this would be seed derived ABA since water deficits were not apparent in BOD with the recent rainfall and high humidity. In contrast, NCED3 and NCED5 had higher transcript abundance in RNO berry skins, nft growing system which might occur as the result of the very low humidity and large vapor pressure deficit .

The lower expression of NCED6 in RNO berry skins may indicate that the seeds in the berry were more immature than the BOD berries. The higher expression of other seed development and dormancy genes in the berry skins support the argument that BOD berries matured at a lower sugar level than the RNO berries. The ABA concentrations in the berry skins are a function of biosynthesis, catabolism, conjugation and transport. ABA in seeds increase as the seed matures and some of this ABA may be transported to the skin. In fact, a number of ABCG40 genes, which encode ABA transporters, had higher transcript abundance in BOD berry skins than that in RNO . Part of the ABA in skins may be transported from the seed and part of it might be derived from biosynthesis in the skins. NCED6 transcript abundance in the skins was higher in BOD berries. Perhaps the transcript abundance of NCED6 in the skin is regulated by the same signals as the embryo and reflects an increase in seed maturity. AtNCED6 transcript abundance is not responsive to water deficit in Arabidopsis, but AtNCED3 and AtNCED5 are. This is consistent with the higher NCED3, NCED5 and BAM1 transcript abundance in RNO berries . Thus, there are complex responses of ABA metabolism and signaling. It would appear that there may be two different ABA pathways affecting ABA concentrations and signaling: one involved with embryo development and one involved with the water status in the skins. Auxin is also involved with ABA signaling during the late stages of embryo development in the seeds. Auxin signaling responses are complex. ABF5 is an auxin receptor that degrades Aux/IAA proteins, which are repressors of ARF transcriptional activity. Thus, a rise in auxin concentration releases Aux/IAA repression of ARF transcription factors, activating auxin signaling. In the berry skins, there was a diversity of transcriptional responses of Aux/IAA and ARF genes in the two locations, some with increased transcript abundance and others with decreased transcript abundance.

As with ABA signaling, there may be multiple auxin signaling pathways operating simultaneously. One pathway appears to involve seed dormancy. ARF2 had a higher transcript abundance in BOD berries. ARF2 promotes dormancy through the ABA signaling pathway. This is consistent with the hypothesis that BOD berries reach maturity at a lower sugar level than RNO berries.Grapevines have very dynamic gene expression responses to pathogens. The top 150 DEGs for BOD berries were highly enriched with biotic stress genes. The BOD vineyard site had a higher rainfall and higher relative humidity than RNO and these conditions are likely to be more suitable for fungi to grow. We detected a much higher transcript abundance of powdery mildew-responsive genes in BOD berries and this may be connected to a higher transcript abundance of ethylene and phenylpropanoid genes as part of a defense response. The transcript abundance profiles of some of these genes are remarkably similar. Increased ethylene signaling in grapevines has been associated with powdery mildew infection and phenylpropanoid metabolism and appears to provide plant protection against the fungus. Genes involved with phenylpropanoid metabolism, especially PAL and STS genes, appear to be quite sensitive to multiple stresses in the environment. In Arabidopsis there are four PAL genes. These PAL genes appear to be involved with flavonoid biosynthesis and pathogen resistance in Arabidopsis. Ten different PAL1 and two PAL2 orthologs had higher transcript abundance in BOD berry skins; many STS genes also had a higher transcript abundance in BOD berry skins . Stilbenes are phytoalexins and provide pathogen resistance in grapes and STS genes are strongly induced by pathogens. Thus, the higher transcript abundance of powdery mildew genes may be associated with the higher transcript abundance of genes in the ethylene and phenylpropanoid pathways.

The transcript abundance of a number of iron homeostasis genes were significantly different in the two locations and there was a difference in soil available iron concentrations in the two locations. However, iron uptake and transport in plants is complicated depending on multiple factors, such as pH, soil redox state, organic matter composition, solubility in the phloem, etc. Thus, it is impossible to predict iron concentrations in the berry without direct measurements. The roles of these genes in iron homeostasis and plant physiological functions are diverse. Iron supply can affect anthocyanin concentrations and the transcript abundance of genes in the phenylpropanoid pathway in Cabernet Sauvignon berry skins. One of the DEGs, SIA1, is located in the chloroplast in Arabidopsis and appears to function in plastoglobule formation and iron homeostasis signaling in concert with ATH13. Another DEG, YSL3, is involved in iron transport. It acts in the SA signaling pathway and appears to be involved in defense responses to pathogens. It also functions in iron transport into seeds. FER1 is one of a family of ferritins found in Arabidopsis. VIT1 and NRAMP3 are vacuolar iron transporters and are also involved in iron storage in seeds. Other DEGs are also responsive to iron supply. IREG3 appears to be involved in iron transport in plastids; its transcript abundance increases with increasing iron concentrations. ABCI8 is an iron-stimulated ATPase located in the chloroplast that functions in iron homeostasis. It is unclear what specific roles these iron homeostasis genes are playing in grape berry skins, but they appear to be involved in iron storage in seeds and protection against oxidative stress responses. One possible explanation for the transcript abundance profiles in the BOD and RNO berry skins is that ferritins are known to bind iron and are thought to reduce the free iron concentrations in the chloroplast, thus, reducing ROS production that is caused by the Fenton reaction. As chloroplasts senesce during berry ripening, iron concentrations mayrise as a result of the catabolism of iron-containing proteins in the thylakoid membranes; thus, berry skins may need higher concentrations of ferritins to keep free iron concentrations low. This might explain the increase in ferritin transcript abundance with increasing sugar levels. Most soils contain 2 to 5% iron including available and unavailable iron; soils with 15 and 25 μg g− 1 of available iron are considered moderate for grapevines, but soils with higher concentrations are not considered toxic. Therefore, vertical hydroponic nft system for both soils in this study, iron concentrations can be considered to be very high but not toxic. The higher available iron concentrations in the BOD vineyard may be associated with the wetter conditions and the lower soil pH.Other researchers using Omics approaches have identified environmental factors that influence grape berry transcript abundance and metabolites. One study investigated the differences in transcript abundance in berries of Corvina in 11 different vineyards within the same region over 3 years. They determined that approximately 18% of the berry transcript abundance was affected by the environment. Phenylpropanoid metabolism was very sensitive to the environment and PAL transcript abundance was associated with STS transcript abundance. In another study of a white grape cultivar, Garganega, berries were analyzed by transcriptomic and metabolomic approaches.

Berries were selected from vineyards at different altitudes and soil types. Again, phenylpropanoid metabolism was strongly influenced by the environment. Carotenoid and terpenoid metabolism were influenced as well. Two studies investigated the grape berry transcriptomes during the ripening phase in two different regions of China, a dry region in Western China and a wet region in Eastern China. These two locations mirror some of the differences in our conditions in our study, namely moisture, light and elevation, although the dry China western region has higher night temperatures and more rainfall than the very dry RNO location. In the Cabernet Sauvignon study, they compared the berry transcriptomes from the two regions at three different stages: pea size, veraison and maturity. The TSS at maturity was slightly below 20°Brix. Similar to our study, the response to stimulus, phenylpropanoid and diterpenoid metabolism GO categories were highly enriched in mature berries between the two locations. Differences in the transcript abundance of NCED and PR proteins were also noted. Like in our study, the authors associated the transcript abundance of these proteins to the dry and wet locations, respectively. In the second study comparing these two regions in China, the effects of the environment on the metabolome and transcriptome of Muscat Blanc à Petits Grains berries were investigated over two seasons; specifically, terpenoid metabolism was targeted. Like in our study, the transcripts in terpenoid were in higher abundance in the wetter location. The transcript abundances were correlated with terpenoid concentrations and a coexpression network was constructed. A specific set of candidate regulatory genes were identified including some terpene synthases , glycosyl transferases and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase . We examined the transcript abundance of some of these candidate genes in our own data but did not find significant differences between our two locations. The contrasting results between our study and Wen et al. could be for a variety of reasons such as different cultivar responses, berry versus skin samples, or different environmental conditions that affect terpenoid production. Terpenoid metabolism is influenced by the microclimate and is involved in plant defense responses to pathogens and insects. Light exposure to Sauvignon Blanc grapes was manipulated by removing adjacent leaves without any detectable differences in berry temperatures. Increased light exposure increased specific carotenoid and terpene concentrations in the berry. The responses of carotenoid and terpenoid production to temperature are less clear. Some effect of temperature was associated with carotenoid and terpenoid production, but to a lesser extent than light. Higher concentrations of rotundone, a sesquiterpene, have been associated with cooler temperatures. Water deficit can also alter carotenoid and terpenoid metabolism in grapes. Terpenes can act as signals for insect attacks and attract insect predators. Thus, terpenoid metabolism is highly sensitive to the environment and influenced by many factors. In contrast to these studies, excess light and heat can affect transcript abundance and damage berry quality. In addition to a higher rate of malate catabolism, anthocyanin concentrations and some of the transcript abundances associated with them are decreased as well.BOD berries reached maturity at a lower °Brix level than RNO berries; the cause is likely to be the warmer days and cooler nights in RNO. Higher day temperature may increase photosynthesis and sugar transport and cooler night temperatures may reduce fruit respiration. °Brix or TSS approximates the % sugar in a berry and is a reliable marker of berry maturity in any given location; however, TSS is an unreliable marker of berry maturity when comparing grapes from very different climates. The differences in TSS between BOD and RNO are consistent with other studies on the temperature effects on berry development. Indirect studies have associated gradual warming over the last century to accelerated phenology and increased sugar concentrations in the grape berries.

The Berry phase reveals geometric information of quantum wave functions via their phases acquired after an adiabatic cyclic process, and its concept has laid the foundation for understanding many topological properties of materials. The theory of Berry phase is built on pure quantum states. For example, the ground state fits the description as the limit of a statistical ensemble at zero temperature. At finite temperatures, the density matrix describes thermal properties of a quantum system by associating a thermal distribution to all the states of the system. Therefore, it is an important task to generalize the Berry phase to the realm of mixed quantum states. There have been several approaches to address this problem, among which the Uhlmann phase has attracted much attention recently since it has been shown to exhibit topological phase transitions at finite temperatures in several 1D, 2D, and spin-j systems. A key feature of those systems is the discontinuous jumps of the Uhlmann phase at the critical temperatures, signifying the changes of the underlying Uhlmann holonomy as the system traverses a loop in the parameter space. However, due to the complexity of the mathematical structure and physical interpretation, the knowledge of the Uhlmann phase is far less than that of the Berry phase in the literature. Moreover, only a handful of models allow analytical results of the Uhlmann phase to be obtained. The Berry phase is purely geometric in the sense that it does not depend on any dynamical effect during the time evolution of the quantum system of interest . Therefore, the theory of the Berry phase can be constructed in a purely mathematical manner. As a generalization, hydroponic bucket the Uhlmann phase of density matrices was built in an almost parallel way from a mathematical point of view and shares many geometric properties with the Berry phase.

We will first summarize both the Berry and Uhlmann phases using a fiber-bundle language to highlight their geometric properties. Next, we will present the analytic expressions of the Uhlmann phases of bosonic and fermionic coherent states and show that their values approach the corresponding Berry phases as temperature approaches zero. Both types of coherent states are useful in the construction of path integrals of quantum fields. While any number of bosons are allowed in a single state, the Pauli exclusion principle restricts the fermion number of a single state to be zero or one. Therefore, complex numbers are used in the bosonic coherent states while Grassmann numbers are used in the fermionic coherent states. Moreover, the Berry phases of coherent states can be found in the literature, and we summarize the results in Appendix A. Our exact results of the Uhlmann phases of bosonic and fermionic coherent states suggest that they indeed carry geometric information, as expected by the concept of holonomy and analogy to the Berry phase. We will show that the Uhlmann phases of both cases decrease smoothly with temperature without a finite-temperature transition, in contrast to some examples with finite-temperature transitions in previous studies. As temperature drops to zero, the Uhlmann phases of bosonic and fermionic coherent state approach the corresponding Berry phases. Our results of the coherent states, along with earlier observations, suggest the Uhlmann phase reduce to the corresponding Berry phase in the zero-temperature limit.

The correspondence is nontrivial because the Uhlmann phase requires full-rank density matrices, which cannot be satisfied only by the ground state at zero temperature. Moreover, the fiber bundle for density matrices in Uhlmann’s theory is a trivial one, but the fiber bundle for wavevfunctions in the theory of Berry phase needs not be trivial. A similar question on why the Uhlmann phase agrees with the Berry phase in certain systems as temperature approaches zero was asked in Ref. without an answer. In the last part of the paper, we present a detailed analysis of the Uhlmann phase at low temperatures to search for direct relevance with the Berry phase. With the clues from the previous examples, we present a conditional proof of the correspondence by focusing on systems allowing analytic treatments of the path-ordering operations. Before showing the results, we present a brief comparison between the Uhlmann phase and another frequently mentioned geometrical phase for mixed quantum states proposed in Refs. , which was originally introduced for unitary evolution but later extended to non-unitary evolution. This geometrical phase was inspired by a generalization of the Mach-Zehnder interferometry in optics and was named accordingly as the interferometric phase. It has a different formalism with a more intuitive physical picture and has been measured in experiments. In general situations, the interferometric phase can be expressed as the argument of a weighted sum of the Berry phase factors from each individual eigenstate. Thus, its relation to the Berry phase is obvious. However, the concise topological meaning of the interferometric phase is less transparent since it is not directly connected to the holonomy of the underlying bundle as the Uhlmann phase does. The reason has been discussed in a previous comparison between the two geometrical phases. The interferometric phase relies solely on the evolution of the system state while the Uhlmann phase is influenced by the changes of both the system and ancilla, which result in the Uhlmann holonomy. Although Uhlmann’s approach can be cast into a formalism parallel to that of the Berry phase as we will explain shortly, its exact connection to the Berry phase is still unclear. The Uhlmann-Berry correspondence discussed below will offer an insight into this challenging problem. The rest of the paper is organized as follows. In Sec. II, we first present concise frameworks based on geometry for the Berry and Uhlmann phases, using a fiber-bundle language. In Sec. III, we derive the analytic expressions of the Uhlmann phases of bosonic and fermionic coherent states and analyze their temperature dependence. Additionally, the Uhlmann phase of a three-level system is also presented. Importantly, the Uhlmann phases of both types of coherent states and the three-level system are shown to approach the respective Berry phases as temperature approaches zero. In Sec. IV, we propose the generality of the correspondence between the Uhlmann and Berry phases in the zerotemperature limit and give a conditional proof. In Sec. V, we discuss experimental implications and propose a protocol for simulating and measuring the Uhlmann phase of bosonic coherent states. Sec. VI concludes out work. The Berry phases of bosonic and fermionic coherent sates and the special cases with a 1D Hilbert space are summarized in the Appendix.The classical approach to the central limit theorem and the accuracy of approximations for independent random variables rely heavily on Fourier transform methods. However, the use of Fourier methods is highly limited without an independence structure, which makes it far less possible to capture the explicit bounds for the accuracy of approximations. In 1972, Charles Stein introduced a novel technique, now known as Stein’s method, for normal approximation. The method works for both independent and dependent random variables. The method also provides bounds of approximation accuracy. Extensive applications of Stein’s method to obtain uniform and non-uniform Berry–Esseen-type bounds for independent and dependent random variables can be found in, for example, Diaconis , Baldi et al. , Barbour , Dembo and Rinott , Goldstein and Reinert , Chen and Shao , Chatterjee , Nourdin and Peccati and Chen and Fang . In addition to the traditional study of Berry–Esseen bounds, new developments to Stein’s method have triggered a series of research on Cramér-type moderate deviations, stackable planters which address the relative error of two tail probabilities. See, for example, Raič , Chen et al. and Shao and Zhou , among others.

Various extensions of Stein’s idea have been applied to many other probability approximations, most notably to Poisson, Poisson process, compound Poisson, binomial approximations and more recently to multivariate, combinatorial and discretized normal approximations. Stein’s method has also found diverse applications in a wide range of fields, see for example,Arratia et al. , Barbour et al. and Chen . Expositions of Stein’s method and its applications in normal and other distributional approximations can be found in Diaconis and Holmes , Barbour and Chen . We also refer to Chen et al. a thorough coverage of the method’s fundamentals and recent developments in both theory and applications. The paper is organized as follows. In the next section, we give a brief review on recent developments on Stein’s method. In Section 3, we present the main results in this paper, the Berry–Esseen bounds and Cramér type moderate deviations for Studentized nonlinear statistics. Applications to Studentized U-statistics and L-statistics are discussed in Section 4. The proofs of the main results are in Section 5, while other technical proofs are postponed to Appendix.A sea change in Colorado politics has vaulted the Democratic Party to unprecedented majorities in the state legislature and a stranglehold on statewide elected office. Democratic dominance to this degree appeared unlikely at the turn of the century when Republicans held majorities in both chambers of the General Assembly, a 4-2 advantage in the state’s U.S. House delegation, both U.S. Senate seats, and the governorship. In the razor-thin 2000 presidential election, Colorado cast its eight electoral votes for Texas Governor George W. Bush who comfortably carried the state by eight percentage points. Republican preeminence in state and federal electoral politics disappeared in less than a generation as Colorado became a solidly blue state with an adrift Republican party unable to wage competitive statewide campaigns. Near supermajority status in the General Assembly and firm control over all statewide executive offices has positioned Colorado Democrats with exceptional political power. While the negative economic effects of the COVID-19 pandemic continue to linger, the Colorado economy has generally rebounded from the great upheaval more rapidly than most other states . Economic growth has propelled increases in revenue; however, the Taxpayer’s Bill of Rights imposes substantial constraints on the total amount of funds available for policymakers to distribute. Spending commitments, such as mandatory increases in K-12 education funding as required by Amendment 23, further cut into the total amount available to appropriate, which creates difficult choices for members of the Joint Budget Committee. Ratified into Article X, Section 20 of the state constitution by voters in 1992, TABOR imposes restrictions on both revenue and spending. Because TABOR limits revenue collections to the prior year’s amount plus population growth and inflation, Colorado taxpayers have received $8.2 billion in TABOR refunds since its enactment including $525.5 million in 2021 and a record $3.7 billion in tax refunds in 2022 . Although it is difficult to amend the state constitution, Colorado voters have considered ballot measures proposing TABOR reform in nearly every election cycle since its adoption. Few have succeeded. The approval of just 11 of the 36 ballot measures to amend TABOR corresponds to a failure rate of nearly 70% . As a result of this unsuccessful track record to modify or repeal TABOR, its shadow continues to loom large over budgetary politics in the Centennial state. An exception to the general inability of reformers to modify TABOR occurred in 2005 when voters narrowly approved referendum C with 52% voting yes. This notable exemption to TABOR permitted the state to spend all revenue collected across the next five fiscal years, which resulted in nearly $3.6 billion in spending that would have otherwise returned to taxpayers during this time frame . Beginning in fiscal year 2010, referendum C permits the General Assembly to retain and spend all funds collected up to the “Referendum C cap.” The passage of referendum C provided greater opportunities for financial investment in areas such as health care, education, and transportation, as well as greater support for police, fire fighters, and other first responders. In retrospect, the successful passage of this reform in an off-year election was anomalistic as voters have since rejected several ballot measures to modify TABOR spending limits . Voters have also opposed an array of tax increases on a dozen occasions including proposals to fund public schools and transportation . Sensing an opportunity to capitalize on the public’s desire for property tax relief, Democrats unsuccessfully sought to connect a reduction in the property tax rate with further erosion of TABOR in 2023. Despite slowing population growth, property values across Colorado continue to soar.