To test whether wind could carry pollen grains through the mesh, we conducted the following experiment. An almond branch with more than 50 flowers whose anthers were dehiscing was held between an electric fan and a new, square plastic plant pot unused cage free from pollen grain contamination. Inside the cage four microscope slides were placed at the same height as the flowers, to intercept any pollen grains that might have passed through the mesh. No pollen packets or single pollen grains of almond could be detected with light microscopy on the microscope slides, although using the same technique without a cage many pollen grains were caught. Cages were removed after blooming was completed, just before trees began to develop leaves. In winter trees were not irrigated and fertilised. The experimental water and nutrient treatments were conducted from January to August 2008. The following nutrients were applied every month by hand when irrigated: 521.6 g nitrate, 344.7 g potassium, 244.9 g sulphur, 158.8 g calcium, 158.8 g phosphorus, 54.4 g magnesium, 27.22 g boron, 27.22 g iron, 27.22 g manganese, and various micronutrients including zinc, cobalt, molybdenum . No nutrients were applied to trees in the no nutrient treatment. Water reduction of the typical irrigated volume for this region and age of the trees was accomplished by manipulating the irrigation system of tubing and emitters at each tree. For the water reduction regime, three out of the four emitters at each tree were closed, reducing water to 27 l every third day. The fungicide Rovral was applied at the rate of 0.0844 g m 2 before rain during blooming to avoid fruit fungal infections.

To quantify fruit set at different developmental stages, we counted the total number of withered flowers on each main branch of each experimental tree from 28 February to 10 March, and we then counted developing fruits four times every 3–4 weeks . On 2 July, we harvested and counted all fruits per whole tree for the last time and then kept 48 fruits per tree in the lab for further measurements. Fruits were randomly selected from the main branches . Freshly harvested fruits were dried on the ground for 7 days while protected from bird and mammal predation with metal cages. After fruit drying, the hulls were removed and shells cracked. We characterised kernel quality by counting the number of unfilled, single and double kernels and the number of kernels damaged by arthropod pests or fungal and bacterial diseases. We measured the length and weight of each of the 48 kernels per tree. On the same dates as developing fruits were counted, we counted the number of leaves, starting at the tip of the main branches for 20 cm and noted the length and colour of ten randomly selected leaves per main branch of each tree. Leaf loss was calculated as the proportion of leaves that dropped between full development of the leaves and fruit harvest .The effect of the treatments on the following response variables were analysed: fruit set and its decrease over time , estimated total number of harvested kernels, mean kernel weight based on the 48 kernels per experimental tree harvested for detailed measurements, and estimated total yield per tree at harvest . To quantify the vegetative response to treatments, we also analysed effects on the number of leaves, proportion of leaves lost from 4 weeks after blooming until harvest, and the proportion of yellowing leaves.

Fruit set over time was modelled using generalised linear mixed models with a binomial distribution and a logit link. We accounted for non-independence of multiple measurements per tree and for extra-binomial variance by including tree and observation, respectively, as a random factor in analyses. Total number of harvested kernels, mean kernel weight and yield were analysed for differences among pollination and resource treatments using generalised linear models . The number of harvested kernels and yield were lntransformed to reduce variance heterogeneity. For analyses of number of kernels and yield, the number of flowers was included as a covariate in the models, since this is a pre-treatment variable that varies from tree to tree . The ln-transformed number of flowers was centred on its mean to make model interpretation easier. For analysis of mean kernel weight, the number of harvested kernels was included as a covariate. Treatment effects on number of leaves, the proportion of leaves lost and the proportion of yellow leaves were analysed using GLM. Average number of leaves per branch was analysed using a GLM for normal data, with the response variable untransformed. Leaf loss was analysed with a GLM for binomial data as a proportional variable. A quasi-binomial GLM was used to model the tree-level leaf colour outcome, identified as the most frequent leaf colour recorded on the tree, with a binary variable . We removed interactions that did not contribute at least marginally to the model . Non-significant main effects were retained. For individual variables, F and P-values in the text are from comparisons between the model with all main effects and significant interactions and the model with the tested variable dropped. All analyses were performed using R, version 2.8.1 for Windows . Mixed models were fit using lmer .Our experiment shows that pollination strongly limits almond fruit set and yield and therefore supports general expectations and previous results of high pollinator dependency in almond . The strong pollination effect on yield even in conditions of reduced water input and nutrient reduction was in contrast to descriptions of California almond production as dependent on high water and nutrient inputs . The negative effects of water reduction on yield, with only marginal negative effects on fruit set and mean kernel weight and no detectable effect on the number of kernels, in this study is supported by previous studies that showed negative effects of water stress on yield , but not on bud development, 25 liter square pot fruit abortion and kernel weight . Surprisingly, the initial benefit of pollination on yield components was not eliminated by reduced water and was not offset by the negative relationship between number and weight of kernels. Although leaf water potential was not measured in this study, as in other work , water stress was indicated as increased leaf loss occurring in the reduced water treatment. Such leaf loss is often observed in water-limited almond trees . The strong effect of reduced water on leaf loss, its marginal effect on mean kernel weight and the increased number of yellowed leaves in open- and hand-pollinated trees with reduced water indicate that when under water stress, almond trees may allocate resources selectively to maintain kernel quantity while reducing kernel quality and delivery of resources to leaves. The lack of any direct significant effects of the cut-off of nutrients on fruit set, yield or leaf loss suggests that the young trees may have already accumulated sufficient nutrients for fruit maturation from the previous summer’s nutrient applications. Nevertheless, the significantly higher proportion of yellowed leaves at harvest on trees receiving no nutrients and reduced water, and the significant interaction between the water and nutrient treatments on leaf colour indicate that the trees were stressed in this treatment combination, especially when pollination took place.

Trees from which pollinators were excluded were characterised by canopies consisting of dense, large and dark-green leaves, in contrast to hand-pollinated trees characterised by small, yellow-green leaves. These differences in foliage indicate that excess nutrients beyond those needed for nut production in the pollinator-excluded trees were used for canopy development. Thus, the positive effect of pollination on fruit production comes at the expense of vegetative performance features and may have long-term consequences for the tree. We found a significant interaction of pollination and irrigation on yield resulting from decreased yield in hand- and open-pollinated treatments receiving reduced water, but no effect of reduced water on yield in the pollinator exclusion treatment, indicating a threshold of pollination is needed before the negative relationship between pollination quantity and water reduction on yield manifests itself. Two other studies analysed the interactions between pollination and plant resources on fruit set in woody plants . Niesenbaum focused, in two consecutive years, on a dioecious, understorey forest shrub whose reproduction was highly limited by light, but not by pollination, with no interaction effect between pollination and light. In contrast, Groeneveld et al. manipulated pollination, light, nutrient and water input and tested for the single and interaction effects of these variables on fruit set and number of harvested cacao pods after 1 year. They found that shade increased the number of aborted fruits, and the interaction of hand-pollination with shade, as well as the interaction of hand pollination with nutrients, reduced the number of fruit abortions, but the interaction effects were not translated to losses or increases in fruit set or yield found in our study. To our knowledge, the present study is the first in which significant interactions between pollination and plant resources on fruit set and yield were found, highlighting the importance of studying pollination and plant resources in a full factorial design to understand their single and combined effects on plant performance in general and crop production in particular. Almond yield was extremely low when pollinators were excluded, although these trees produced large kernels, while yield of hand-pollinated trees were high with small kernels. The kernel size in the different pollination treatments is likely caused by resource allocation and availability rather than pollination quality. In the pollinator exclusion treatment, kernels are assumed to result from self-pollination with low quality and quantity pollen. These results are contrary to studies showing that fruit or seed size and weight are often positively related to pollination quality and quantity . It also indicates that intensive pollination management, such as simulated by our hand-pollination treatment, can result in low kernel quality . Future experiments conducted over consecutive years are needed, particularly because high fruit set in year one resulting from supplemental pollination in the previous year may impose limits on reproduction in subsequent years . We found that foliage was reduced by water stress and indirectly by pollination in our 1-year study, but this may influence fruit set in the following year because the number and size of leaves influences rates of photosynthesis and hence resources available to develop new flowers . Further, fruit load may be more strongly determined by the stress history of the trees rather than the current year’s irrigation treatments . Although the need to study pollination and resource limitation for several years in perennial plants is evident, the pollinator-dependent yield response determined with and without resource limitation of a single year can help growers to make ad-hoc decisions in years of pollinator and/or water shortages. Our results suggest that for almond, pollination of the crop should be a high priority, but that other resources must be concurrently monitored and managed because of their well known effects and potential interactions that can influence overall plant performance.

Gene regulation of host plant expansion has been revealed in several herbivorous insects, including Subpsaltria yangi Chen , Drosophila mettleri Heed , and Chilo suppressalis Walker . For example, research on host plant expansion in a cactophilic fy, Drosophila mojavensis , revealed cytochrome P450, glutathione S-transferases, and UDPglycosyl transferases as major gene classes involved in new host use . There has been limited research on the genetic mechanisms of host plant expansion in tephritids. Therefore, the present review summarizes current knowledge on the categories and roles of the genes involved in host plant expansion in tephritids and the related regulatory mechanisms and relates these fndings to the development of new control methods for tephritid species.Volatile chemicals stimulate chemosensory receptors in tephritid flies when assessing a potential novel host and trying to expand . Therefore, chemosensory-related genes are involved in the initial process of host plant expansion for tephritids. Olfactory-related genes of tephritids are one type of chemosensory gene that includes several gene families of odorant-binding proteins , chemosensory proteins , odorant receptors , ionotropic receptors , and sensory neuron membrane proteins , which are primarily involved in the identifcation of volatile chemicals, including volatiles of host fruits. After receiving odor chemical signals, these olfactory-related genes are triggered to transduce cascades that send information to specific regions of the brain, which ultimately leads to specific behavioral responses .

OBP genes play an important role in the first step of chemosensory identification of insects, including tephritids . OBP genes direct odorant-binding proteins to bind volatile odor molecules specifically by distinct expression to related olfactory receptors that are bound to olfactory receptor neurons in antennae . CSP genes are regarded as playing a similar role as OBP genes involved in the initial process of chemosensory signal transmission to corresponding receptors . OBP and CSP genes are major gene types that lead tephritid flies to respond to different chemosensory chemicals, including volatile chemicals of host plants . Except for these two categories of genes, some odor receptor genes also play important roles in host odor recognition of tephritids, such as genes related to odor receptors and ionotropic receptors . Odorant receptors of insects are composed of at least two proteins: a conserved coreceptor as an ion channel and a specific OR subunit , which determines the ligand specificity and forms structurally ligand-gated ion channels . The OR genes mediate odorant receptors of insects transmitting the odorant molecules they receive into electric signals that are transmitted to a higher-order neural center . IR genes are related to ionotropic glutamate receptors , which are regarded as ion channels . They also play important roles in odor chemical perception . The sensory neuron membrane proteins gene encodes transmembrane domain-containing proteins that belong to a large gene family of CD36 receptors . SNMPs regulates the corresponding proteins to identify chemosensory signals, mainly pheromone chemicals .

The GR family is another type of chemosensory protein that is a ligand-gated ion channel broadly expressed in gustatory receptor neurons in taste organs and is mainly involved in taste recognition of CO2 , sugar, and bitterness . When receiving taste signals, GR genes are involved in identifying taste and ingestion. Among tephritid flies, Bactrocera dorsalis and Ceratitis capitata are well-known polyphagous species that have expanded their host plants to more than 250 species . However, Bactrocera minax and Z. cucurbitae are oligophagous species that mainly attack citrus fruits and cucurbit plants, respectively. Bactrocera oleae , Procecidochares utilis , and Carpomya vesuviana  are monophagous species infesting olive , crofton weed , and jujube , respectively, and all have limited host plant species . Compared to several major olfactory-related gene families, the two polyphagous species have more genes, with 3 CSPs, 35 OBPs, 74 ORs, and 40 IRs in B. dorsalis and 45 OBPs, 76 ORs, and 70 IRs in C. capitata , than two host-limted species . A similar situation was observed in the GR family. There are also more GR genes in C. capitata and B. dorsalis than in the host-limited species P. utilis , C. vesuviana , and Z. cucurbitae . The increased numbers of these genes are associated with chemosensory-related gene family expansion via gene duplication and differentiation , which exertimportant roles in tephritid fy adaptation to other hosts and expansion of their host ranges. Obvious chemosensory-related gene expansions were also reported in Tribolium castaneum , Spodoptera frugiperda , and Heliconius melpomene . For example, the pea aphid Acyrthosiphon pisum , with broader host ranges, experienced obvious expansion of the OR, OBP, and GR gene families, with 87 ORs, 18 OBPs, and 78 GRs, compared to the soybean aphid Aphis glycines , with 47 ORs, 10 OBPs, and 61 GRs . Altering gene expression levels also helps tephritids respond to different host plants and realize host expansion. OR13a and OR82 expression are higher in antennae in B. dorsalis in response to 1-octen-3-ol and geranyl acetate, respectively, which are major volatile components of its host fruits, mango and almond fruit . For B. minax, increasing the expression levels of several GR genes regulate the taste process in response to different chemosensory stimuli of hosts .Once a tephritid adult identifes a potential novel host fruit for oviposition or feeding, the plant fruit must be suitable for larval development, which includes overcoming any secondary toxic chemicals in the novel host fruit . Therefore, detoxifcationand other digestion-related genes also play core roles in mediating the host plant expansion of tephritids. Common detoxifcation-related genes of insects include gene families of cytochrome P450s , glutathione S-transferases , UDP-glycosyltransferases , carboxyl/cholinesterases and ATP binding cassettes . The cytochrome P450 family belonging to phase I enzymes includes various CYP subfamilies for different tephritid species . The GST superfamily consists of phase II enzymes divided into at least seven major subclasses: the delta, epsilon, omega, sigma, theta, zeta, and microsomal classes . The PGE phase II enzymes are a large family that can be divided into 13 clades, including the dietary detoxification class , black plastic planting pots the hormone/semiochemical processing group , and the neurodevelopmental group . The ABC transporter superfamily belonging to phase III enzymes can be subdivided into eight subfamilies, from ABC-A to ABC-H. The cytochrome p450 gene family of phase I mainly contributes to the catalysis of numerous oxidative reactions during endogenous and exogenous metabolism . The important roles of genes in this family are the metabolism of xenobiotics, plant allelochemicals , and even insecticides. GSTs are multifunctional genes of phase II enzymes that play a crucial role in the detoxifcation of endogenous and xenobiotic compounds, including plant secondary metabolites and pesticides. CCE families of phase II have been shown to be involved in the detoxifcation of plant-derived allelochemicals as well as insecticides . The ABC transporter genes of phase III encoding membrane-bound proteins typically function in the ATP-dependent transport of various substrates across biological membranes . The roles of ABC genes are mainly in handling xenobiotics such as plant phytotoxins and insecticides .

These genes can participate in regulating detoxifcation of host plant secondary metabolites of tephritid flies by coding corresponding enzymes, which help to transform toxins entering the insect system into hydrophilic compounds that can be eliminated and in the adaptability of different hosts . The major digestive-related genes include gene families of cysteine proteases, proteases, lipase, glucosidase, and serine proteases . The serine proteases are members of the supergene family, including chymotrypsin, trypsin, thrombin, subtilisin, plasmin, and elastase. subclasses . Various digestive proteases exert important roles in the nutrition digestion of tephritid flies from novel host plants that they try to expand to. However, protease inhibitors of host plants are a widespread defense against herbivores such as tephritids. Therefore, genes coding various proteases react to protease inhibitors by regulating inhibitor-sensitive proteases or expressing proteases that are not targets of the inhibitors . When expanding to other novel hosts, tephritid flies must adapt to different chemical environments from their native hosts. Detoxifcation-related genes regulate the host expansion of tephritids via gene family expansion similar to chemosensory-related genes. The major gene families of detoxifcation GSTs, P450s, CCEs and ABC transporters are more numerous in polyphagous B. dorsalis  and C. capitata than monophagous P. utilis and B. oleae . However, reports about digestive gene family expansion in tephritids are still rare. Overall, detoxifcation and the digestive-related gene family combined with chemosensory-related gene family amplifcation exhibit a close association with host range extension. This gene family expansion is helpful for the host plant expansion of fruit flies. Cases in other insects strengthen this idea. For example, Helicoverpa armigera and Helicoverpa zea are two species of caterpillars that have considerably broader host ranges than any other lepidopterans. Great expansion of detoxifcation and digestive gene families was found in the two species. In addition to gene family amplifcation, detoxifcation and digestive genes also regulate host expansion of tephritid flies by activating various gene subfamilies, subclasses, or clades. To respond to various toxic environments, including secondary toxic chemicals of different hosts, B. dorsalis primarily triggered the delta subfamily of GSTs, CYP3 and CYP4 subclasses of P450s, A–C clades of CCEs, and ABC-A, ABC-B, and ABC-G subclasses of ABC transporters , C. ceratitis activated the epsilon subfamily of GST, CYP6 and CYP12 of P450s, B clade of CCEs , and P. utilis mainly triggered the delta, epsilon and microsomal subfamilies of GSTs, CYP4, and CYP9 of P450s, C clade of CCEs and ABC-G subclass of ABC transporters , but R. pomonella mainly launched CYP4 and CYP6 of P450s . For the digestive gene family, B. dorsalis and C. capitata primarily triggered aminopeptidase, trypsin and serine peptidase digestive genes, but B. oleae, which is a strictly monophagous species, triggered serine protease and nuclease digestive genes to respond to different host secondary chemical environments . Detoxifcation- or digestion-related genes also facilitate tephritid fy adaptation to different hosts by altering gene expression levels. Rhagoletis zephyria evolved fromRhagoletis pomonella and experienced host expansion from apple to snowberry plants . Increased expression levels were found in some detoxifcation-related genes, including cytochrome P450, glutathione S-transferases, and glycosyltransferase, in R. zephyria facing the apple host environment . Z. cucurbitae is the species that mainly attacks cucurbit plants, and the fy responds to different secondary chemical environments of Mucuna pruriens plants by reducing the expression levels of trypsin and chymotrypsin digestive genes .Although the importance of chemical stimuli is highly emphasized in the host expansion of tephritids, other nonchemical stimuli, such as the color of the novel host fruit, should not be ignored. Many insects locate their host plants primarily by color signals, including beetles, Altica engstroemi , Hylastes ater , and Arhopalus ferus . For tephritid flies, Neoceratitis cyanescens , B. minax , B. dorsalis , and Z. cucurbitae are typical examples of species that appear to select different hosts first by fruit color rather than chemical signals. Z. cucurbitae realized its host expansion to a novel host, papaya , in Hawaii by strongly relying on the color location of fruits by vision .

The accumulation of sucrose per fresh weight peaked in the juice sacs during stage II of fruit development . Sucrose hydrolysis seemed to be mediated by all enzymes, as the activity of SuSy and that of the three forms of invertase were detected in the juice sacs . However, most studies showed that the activity of vacuolar invertase was relatively high, followed by SuSy activity. The activity of cell-wall invertase was also detected, but at a lower level, and soluble invertase activity was lowest. The presence of plasmodesmata has so far not been demonstrated, and cell-to cell movement might also follow a symplasmic pathway. Considering the relatively high activity of the vacuolar invertase, temporal storage and compartmentalization of sugars should occur during transport. Moreover, as the activity of cell wall invertase was also demonstrated, apoplasmic movement cannot be ruled out, and it might also play a role in temporal storage. Lowell et al. indicated that young fruit might behave differently than mature ones, as the former displayed uphill transport in terms of sugar concentration whereas fully grown fruit displayed downhill transport .

Interestingly, out of the six SuSy genes in the citrus genome, two were induced in juice sacs during development, with one of them induced in the segment epidermis as well, suggesting that SuSy acts in sucrose mobilization within the juice sacs . As expected, invertase activity in all cellular compartments was reduced toward fruit maturation, in good correlation with the reduction in the invertase transcripts . The activity and transcript levels of sucrose phosphate synthase genes were induced in Satsuma fruit juice sacs toward maturation, in accordance with an increase in sucrose level; however, in grapefruit, enzyme activity was induced from stage I to stage II of fruit development, and decreased toward maturation . This might explain the difference in sucrose levels between the two cultivars, as grapefruit accumulates less sucrose than Satsuma mandarin. Sucrose phosphate phosphatase was also induced during later stages of fruit development, suggesting that sucrose accumulation did not result only from translocation from the leaves but also from active synthesis within the juice sac cells . Nonutilized sucrose is stored in the vacuole and therefore, sucrose transport across the tonoplast might well play a role in regulating its levels within the cell and even its unloading rate. Sucrose and hexose uptake into tonoplast vesicles of sweet lime was not induced by ATP, suggesting facilitated diffusion .

Inclusion of acid invertase protein in the vesicles induced sucrose uptake, suggesting that sucrose hydrolysis by invertase or chemical acid hydrolysis within the vacuole provided the driving force for its uptake . An endocytic mechanism for sucrose transport across the tonoplast was also suggested .While being transported into the fruit, sucrose can undergo metabolism in a few directions. Hexose phosphate synthesis is an important metabolic step, with the reversible conversion of fructose-1-phosphate and fructose-1,6-biphosphate providing a link between sugar and organic metabolism via glycolysis/gluconeogenesis pathways . The reaction is catalyzed by two independent mechanisms . One involves two enzymes, an ATP-dependent phosphofructokinase catalyzing the glycolytic conversion of Fru-6-P to Fru-1,6-P2, and fructose-1,6-bisphosphatase , catalyzing the reverse, gluconeogenic reaction. The other mechanism is composed of one bidirectional enzyme, pyrophosphate-dependent PFK composed of two subunits, PFPα and PFPβ . Whereas PFK is generally considered ubiquitous, PFP has been described in prokaryotes and lower eukaryotes, including some bacteria, and some protozoan parasites . In addition, it is found in higher plants, where it is expressed in various tissues . While plants contain both PFP and PFK, bacteria and protozoa appear to have either one or the other, and yeast and animals contain only the latter . PFK is considered the more abundant enzyme, but its activity in plants is less characterized than that of PFP, due to its instability upon purification. PFK is found in both the cytosol and the plastids, whereas PFP is a cytosolic enzyme. Several hypotheses have been raised to explain the role of PFP in plants, including activation during stress . Transgenic up/downregulation of PFP in tobacco, potato, and sugarcane resulted in only minor alternations in plant growth and metabolism . However, reduced expression of PFP in Arabidopsis resulted in delayed development, while higher expression resulted in induced development . Moreover, knockout mutants suggested that PFP is required for adaptation to salt and osmotic stress during germination and seedling growth . While Fru-2,6-P2 is the major PFK activator in microorganisms and animals, in plants it does not activate PFK but rather PFP . Citrate was found to be an inhibitor of PFP activity, especially in the glycolytic direction , and was suggested to affect the affinity of Fru-2,6-P2 binding . PFP was detected in the juice sac cells of Valencia orange and grapefruit along with PFK and FBPase . While grapefruit PFP was strongly induced by Fru-2,6-P2 in the forward reaction, it was barely affected by the activator in the reverse reaction , as also demonstrated for potato, pineapple and tomato fruit . It was also shown that citrate, and to some extent other intermediates of the tricarboxylic acid cycle, inhibit the glycolytic reaction of PFP in grapefruit, whereas the gluconeogenic reaction was barely affected . Reduction in PFP activity in the ovaries of open versus closed flowers paralleled the reduction in protein levels of the two subunits, suggesting that the enzyme activity was regulated by its protein levels in the ovary . However, more complex relationships were detected in the fruit, demonstrating the involvement of other mechanisms in regulating PFP activity. Recently, the two subunits of citrus PFP were coexpressed and expressed separately in bacteria . Monomeric forms of both subunits were able to catalyze phosphorylation of Fru-1-P, but when coexpressed, plant pot with drainage the heteromeric form generated activity that was two orders of magnitude larger. While the activity of the heteromeric form was induced by Fru-2,6-P2, that of the β-monomer was repressed and the activity of the α-monomer was barely affected.Pulp acidity in citrus fruit is determined by two separate processes, citrate content in the vacuole of the juice sac celland vacuolar acidification, which can reach 0.3 M and pH 2.0, respectively in lemon and other acidic cultivars . Although separate, these two processes are bioenergetically coregulated . During the first half of fruit development, citrate accumulation is accompanied by proton influx which reduces the vacuolar pH. Citrate has three dissociation constants — 6.39, 4.77 and 3.14 — and in the vacuole it acts as a buffer by binding protons as they accumulate and reducing the pH, thus providing a driving force for additional proton influx . On the other hand, proton influx provides a driving force for citrate uptake, and probably also for its synthesis.

When the vacuolar pH of Navel orange juice sacs was below 3.5, two forms of citrate were detected, citrateH3 and citrateH2 – . CitrateH2− and citrateH3− could be detected in pH ≥ 3.5 and pH ≥ 5.0, respectively. During the second half of fruit development, when the acid level declines, citrate removal is accompanied by proton efflux and increasing pH. There is a good correlation among different citrus cultivars between the level of juice pH and citric acid concentration , and there are no reported cases in which pulp pH and citrate level are both low; therefore, altering citrate concentration will change pH homeostasis, and vice versa. However, early in fruit development, the two processes can be distinguished . Citrate accumulation in Minneola tangelo starts in early June and continues for approximately 3 weeks; during this time, pH is slightly increased, probably due to the dilution effect associated with cell division. Significant pH reduction is only detected after 4 weeks, suggesting that the buildup of some citrate accumulation is required to induce proton influx into the vacuole. This also suggests that citrate accumulation precedes proton accumulation. In other fruit of low and moderate acidity levels, such as melons, i.e., pH 4.5–6.5, some inbred lines with higher pH and higher citrate + malate content than their parents were reported . Although citrate is the major organic acid accumulated in citrus fruit, accounting for 90% of the total acids, the synthesis and accumulation of other organic acids have also been reported . For instance, in orange, there is a transient increase in quinic and oxalic acids early in fruit development. Malic acid also accumulates to some extent during the maturation of lemon, lime and orange fruit.So far, three mechanisms associated with proton movement across the tonoplast have been identified and characterized in citrus juice sac cells : V-type H+-ATPase, the major enzyme driving proton influx; H+-pyrophosphatase; and citrate/H+ symporter, most likely acting to remove citrate−2 out of the vacuole along with 2H+ . Other transport mechanisms, associated with citrate transport across the mitochondrial membrane and citrate movement into the vacuole, have been predicted for other plant species, but not for citrus fruit . A P-type ATPase, homologous to the petunia PH5 and PH8, was suggested to play a role in vacuolar hyperacidification . PH5 and PH5 were recently shown to be highly expressed in acid cultivars and down regulated in acidless cultivars, due to mutations in the MYB, HLH and/or WRKY transcription factors . While PH5 and PH8 were shown to localize to the vacuole in petunia, their membrane localization and biochemical properties in citrus require further research . The identification and characterization of vacuolar transport mechanisms require isolating purified tonoplast vesicles or intact vacuoles . An array of experimental tools can then be used to study transport across the membranes, such as radiolabeled molecules , pH-dependent fluorescent dyes such as acridine orange or quinacrine . An acidic-inside can be generated in isolated tonoplast vesicles or intact vacuoles through the activation of the V-type H+-ATPase or the H+-pyrophosphate with Mg–ATP or Mg– PPi and the use of inhibitors or protonophores to alter the pH gradient. For example, the addition of bafilomycin A inhibits the V-ATPase activity while gramicidin permeabilize the membrane to protons, thus abolishing the DpH across the membrane without affecting the pump hydrolytic activity. Tonoplast vesicles of juice sacs were isolated and purified from acidic cultivars and their acidless counterparts. 14C-citrate uptake of acidless pomelo vesicles was about 20% higher than that of acid pomelo, eliminating the possibility that the difference in fruit acidity between these two cultivars was due to citrate transport into the vacuole . The uptake was enhanced by ATP . Generation of a pH gradient was investigated in tonoplast vesicles of acid and acidless lime. As expected, it was induced by Mg–ATP, while bafilomycin and nitrate inhibited ATP hydrolysis and abolished the pH-gradient formation . Sweet lime tonoplast vesicles appeared to generate a DpH four times faster than those of acid lime, but they had higher H+ leakage following H+-ATPase inhibition by EDTA than the acid lime, possibly representing their limited in-vivo capacity for H+ retention.

The method for FRAP assay is described in detail in Section 3.6. These differences in the FRAP values before and after the encapsulation process reflect the relative amount of antioxidant compounds, including phenolics that are infused or bound to a selected cell-based micro-carrier. Table 1 shows the total antioxidant capacity of MG juice sample measured using the FRAP assay. The encapsulation efficiency in the selected bacterial carrier was 72.67% for MG. This percentage indicates the total fraction of antioxidant compounds bound and encapsulated in a bacterial cell carrier compared to the total antioxidant content in the juice sample. This result suggests that a simple incubation method allows phytochemicals to passively diffuse from a juice matrix to inactivated L. casei cells and results in an efficient binding and encapsulation of the antioxidant compounds in the cell carrier.In addition to characterizing the encapsulated antioxidant content, the encapsulation efficiency of the anthocyanin pigments from the juice to the cells was also evaluated. Anthocyanin, being water soluble, is one of the major polyphenolic fractions in fruit juice and has a significant contribution to its antioxidant properties. To assess the anthocyanin content in the juice before and after encapsulation, the juice matrix was ex-tracted using methanol as described in the materials and methods section and the total anthocyanin content in the extract before and after incubation with cells was measured using a UV-Vis spectrophotometry.

The measured absorbance at 530 nm was converted to an equivalent keracyanin chloride concentration using a standard curve. Results show that MG juice had approximately 8.21 µM/mL of the equivalent keracyanin content. After incubation with inactivated bacterial cells, 66.97% of the total anthocyanin from the MG juice was encapsulated or bound to the cell carriers. In summary, these results highlight a significant potential of the selected bacterial strain for encapsulating antioxidants and anthocyanin family of compounds from a complex juice sample.To help visualize the encapsulated compounds and their intracellular distribution in the cell carriers, confocal multispectral fluorescence images were acquired based on the endogenous fluorescence signals of phytochemicals. The images were collected with a 405 nm excitation and an emission in the FITC channel from 500 to 550 nm. The fluorescence intensity of the cells in each image was quantified by randomly selecting 20 cells and measuring their mean pixel intensity using the ImageJ software. The mean background intensity was subtracted from the cell signals to remove the background signal. As shown in Figure 1, the signal intensity of L. casei carriers increased approximately 24-fold upon incubation of cells with an MG juice as compared to the auto- fluorescence signal from the control cells . Differences in the fluorescence signal intensity between the controls and the modified cells with juice phenolics was statistically significant with a p-value ≤ 0.05. The zoomed-in views in Figure 1b indicated that the cell carriers retained the cellular structure after the encapsulation process, and the encapsulated material was localized relatively uniformly across the intracellular compartment.

The broad emission range is usually associated with the presence of a diverse class of polyphenolic compounds. Based on the previous literature related to fluorescence properties of polyphenolics, the emission band between 533 nm and 595 nm mostly corresponds to anthocyanin content. MG spectra with the secondary emission around 590 nm indicates the presence of anthocyanin compounds in the cell carriers from juice matrix. In addition, the major peak in the MG spectra around 515 nm suggests a possible encapsulation of other phenolic compounds. Plant phenolics such as ferulic acid are known to have fluorescence emissions centered around 520 nm–530 nm. The broadening of the peaks observed in Figure 2 could be attributed to other photo active compounds present in the complex juice matrix. The shift in the emission range compared to the peaks observed from prior literature could also be caused by multiple factors. Anthocyanin polymerization during the juice processing and storage process could cause the emission to shift towards shorter wavelengths. In addition, fluorescence emission spectrums are known to be sensitive to environmental factors, including the excitation wavelength, medium pH and polarity, present macromolecules, etc.. Thus, it may contribute to the shifts observed in Figure 2. In this measurement, phenolic compounds that emit blue fluorescence were not captured in Figure 2 due to the limitation of the available wavelength range in this imaging system. To address these gaps in the compositional analysis of encapsulated compounds, analytical measurements using a HPLC method with known standards were conducted.Among the diverse groups of bio-active compounds present in the fruit and fruit juices, phenolic compounds constitute one of the largest and most diverse groups of phytochemicals. To characterize the phenolic compounds profile of the juice and the encapsulation efficiency, the MG juice before and after incubation with cells was analyzed using HPLC and, based on these measurements, the encapsulation efficiency of the selected polyphenolics was quantified. The protocols for the evaluation of phenolics in a grape juice matrix were already developed by Oberholster et al.. Target compound classes included in this study were flavanols , phenolic acid , flavonols , and polymeric polyphenols . Catechin, epicatechin, gallic acid, and polymeric phenols were quantified using chromatograms at signal 280 nm, caffeic acid at 320 nm, and quercitin and glycosylated myricetin at 360 nm. These polyphenolic compounds have been determined to be among the leading polyphenolic compounds in a grape juice. The chromatograms of 20% MG juice matrix at signals 280, 320, and 360 nmbefore and after encapsulation are shown in Figure 3. Peaks corresponding to each analyzed phenolic compounds were assigned in chromatograms collected at each signal.As observed from Table 2 and Figure 3, most of the investigated compounds were present in MG juice at different concentrations and had different levels of encapsulation efficiency. For flavanols, catechin and epicatechin were both present in the MG juice matrix. Despite these differences in absolute concentration levels, 17.40% of catechin and 18.77% of epicatechin were encapsulated upon incubation of cells with MG juice. For the phenolic acids, large pot with drainage the concentration of gallic acid in MG juice was 1.69 mg/mL and its encapsulation efficiency was 18.43% in L. casei cells. The content and encapsulation efficiency for gallic acid was significantly higher than the amount of coutaric acid and its encapsulation efficiency in L. casei cells. The amount of caffeic acid was below the detection limit in MG juice. Among flavonols, 20% MG juice contains 21.70 mg/mL of quercetin. The encapsulation efficiency of quercetin in L. casei was limited as compared to the quantified flavanols and phenolic acids. Glucoside derivatives are commonly found in grapes and wines, particularly delphinidin-3-glucoside, petunidin-3-glucoside, and malvidin-3-glucoside . MG juice contains 3.53 mg/mL myricetin 3-glycoside and its encapsulation efficiency was 69.85% upon incubation of cells with MG juice. Another common abundant polyphenolic compound in MG juice was polymeric phenols. The 20% juice contains 26.09 mg/mL of polymeric phenols.

The polymeric phenols identified using this protocol represent a mixture of polymeric pigments, which are formed based on reactions between grape anthocyanins and other components in the juice such as tannin, catechins, and proanthocyanidins. A total of 97.97% of the polymeric phenol was infused into the cell carriers upon incubation with MG juice. Taken together, the imaging and HPLC measurement results illustrate that cell carriers can simultaneously encapsulate diversity of bio-active compounds from a complex juice matrix. Compared to previous studies that have predominantly focused on yeast cells for the encapsulation of purified hydrophobic polyphenolic compounds, the results of this study suggest a potential of diverse cell carriers, including bacterial cell carriers, to simultaneously encapsulate multiple compounds from mixtures. Furthermore, since the encapsulation process was conducted using water soluble compounds in fruit juice, this study demonstrates that bacterial cell carriers can bind and encapsulate compounds from water extracts and juices. Together with prior studies, the results of this study illustrate the potential of cell carriers to encapsulate both hydrophobic and hydrophilic bio-actives. The encapsulation process of these compounds from cell carriers can be attributed to both composition and structure of cell carriers. Besides the structural integrity that withstood the encapsulation process as shown in Figure 1, bacterial and yeast cell carriers have a relatively high fraction of protein content on a dry basis. In the case of L. casei cells, the protein content can be as high as 80% or higher on a dry basis. Similarly, the protein content in yeast cells can range from 25 to 60% on a dry basis. In addition, cell carriers also express both soluble and structural proteins including membrane associated proteins. In previous studies, protein–polyphenolic interactions have been explored and the binding between protein isolates and polyphenolic compounds from juice or other plant extracts has been demonstrated. Thus, it is likely that a relatively high concentration and diversity of proteins in micro-scale cells carriers significantly promote the binding of diverse polyphenols from a juice matrix. In addition to proteins, bacteria and yeast cells also contain a diversity of carbohydrate bio-polymers mostly concentrated in cell walls and lipids that are integral parts of the cell membranes. Prior studies have shown interactions between polyphenols and cellular polysaccharides, and the binding mechanism could be attributed to a range of physical and chemical interactions. The complex and porous structures and surface properties of the cell wall has also been proposed to be important for the binding process. These compositions and cellular structures can provide a rich environment for the partitioning and compartmentalization of diverse compounds in cell-based carriers.The results illustrate that the encapsulated content and efficiencies varied by the chemical class, compounds, and juice matrix. Quercetin as a monomeric flavonol showed low incorporation rates from MG juice, while the glycosylated myricetin has a significantly higher encapsulation efficiency . In contrast, polymeric polyphenols yielded the highest encapsulation efficiency among all compounds tested from MG juice . This trend of differences in encapsulation efficiency of compounds of the same class was also observed in the case of flavonols and phenolic acids. Furthermore, based on these measurements, no clear correlation between encapsulation efficiency and relative hydrophilicity of the compounds was observed. These observations suggest that the partitioning of compounds in cells from a juice matrix significantly depends on the interactions among the polyphenolic compounds and the composition of cells. The characterization of these interactions is beyond the scope of this study, but these results suggest that it may be possible to select cellular compositions among the diverse class of microbes that may promote the binding of selective polyphenols from a given plant extract and juice.One of the important functionalities of encapsulation carriers is to protect the bio-active compounds from adverse environmental factors and food processing conditions. These adverse conditions may cause damage by oxidation, less favorable pH, and thermal induced reactions in bio-active compounds in food matrices. In order to produce a shelf-stable and microbially safe food, thermal processing methods such as pasteurization or sterilization are commonly used. Thus, in this study the effectiveness of the selected bacterial carrier in protecting and stabilizing the encapsulated juice polyphenols was evaluated.

Fruit size is among the most crucial fruit quality traits for mandarin breeding. Generally, mandarin fruit size varies from small to medium, but tangelo and tangor hybrids have larger fruit sizes. Amparo is a small-fruited mandarin with about 40 mm diameter and 30 g weight. Moreover, Ugly has a much larger fruit size, it is a tangelo with a fruit diameter of 120 mm and a fruit weight of 580 g . Obtaining fruits in uniform fruit size is one of the essential elements for mandarin breeding. Therefore, it is important to construct a mandarin genetic map and identify markers related to fruit size. QTL identification associated with fruit size for mandarins has been conducted in several studies . In the study, Fortune , Murcott , and 116 F1 mandarin individuals derived from were analyzed for fruit quality traits by Yu et. al . It was carried out in Fortune , Murcott , and 116 F1 mandarin individuals derived from were analyzed for fruit quality traits by Yu et. al . It was carried out in January and February, with four samplings in 2012 and 2013. The map was performed by using a 1536-SNP Illumina GoldenGate assay. The constructed genetic linkage map of “FOR” consisted of 189 SNPs, while “MUR” consisted of 106 SNPs. A total of 48 QTLs related to fruit quality traits were defined in the study. 3 QTLs of them were associated with fruit weight and 3 QTLs of them were related to fruit diameter . The repeatable QTLs were determined as FW5.1 and FW8 and non-repeatable were FW4.2, FD4.2, FD5.1, and FD9.3.

FW4.2 was detected on MUR4.2 with 25.69 cM and explained % 24.60 of the phenotypic variance . QTL analysis study on fruit quality characteristics in the mandarins detected QTLs associated with fruit size. A SNP-based genetic linkage map and QTL mapping were conducted using an F1 segregating population derived from by Imai et. al . The map for “Harehime” consisted of 442 SNPs, and for “Yoshida” consisted of 332 SNPs. 4 QTLs were identified for fruit weight with 14.9-26.5 % of the phenotypic variance. FWq3 was identified spanning 15.3-31.0 cM on the Clementine genome scaffold 4. The most striking point in this study is the claim that these two QTLs can correspond since FWq3 and FW4.2 are located on the same Clementine reference genome scaffold 4 .The fruit flavor is one of the main determinants of fruit quality and consumer preference in mandarins. The ratio between sugar and acid content, which is defined as the total soluble solids: titratable acidity ratio , is significant for the taste of the fruit . Only the high sugar content and less acidity rate or less sugar and high acidity level do not affect the fruit taste positively. For this reason, fruit is required to contain a certain amount of acid content in terms of fruit taste. Fruits with a high TSS/TA ratio have a bland taste, while fruits with a low TSS/TA ratio have a sour taste. Goldenberg et. al stated that the TSS/TA ratio for mandarins that are highly desired is about 13. In addition, Citrus fruit development in relation to solids and acids. Solids gradually increase, and acids first increase and then decrease.

So meaningful comparison of varieties is only possible in relation to the date sampled. reported that tangerines, which is a popular variety in terms of their taste, are the variety of mandarin with mean of the highest sugar content , acid content , and sugar/acid ratios in Table 2. Some QTLs associated with the fruit sugar content in Citrus have been identified in some previous studies. For Soluble Sugar Content , Yu et. al detected five non-repeatable QTLs on scaffolds 2,3,4, and 8 of the Clementine reference genomes. In addition, three non-repeatable QTLs for Soluble solids content: titratable acidity were identified by Yu et. al . Moreover, they identified two non-repeatable QTLs and one repeatableQTL for TA were positioned at scaffolds 7, 8, and 9 of the Clementine reference genome. The QTLs identified for ST were positioned on the scaffold 1, 7, and 9 of the Clementine reference genome . In this study, some QTLs for TA overlapped the QTLs for ST and SSC. For example, TA9 and ST9, and TA8 and SSC8 overlapped. Imai et. al studied one of four mandarin fruit quality traits: sugar content . Only SCq1 was identified for SC on LG5 .Seedlessness is a desirable trait for Citrus breeding since the seed in the fruit may negatively affect the taste and aroma of the fruit due to effects of seeds on chemical composition . Navel orange, Satsuma mandarin, and Clementine mandarin are the most popular Citrus crops that are seedless . For Citrus fruit to be defined as seedless, it must contain no seeds, contain aborted seeds, or the number of seeds of a multi-seeded variety must be significantly reduced . Many factors play a role in mandarin seedlessness, including parthenocarpy, male and female sterility, self-incompatibility, abnormal ovules, embryo sac abortion, environmental conditions, and plant growth regulators . Seedlessness is one of the most important fruit quality characteristics in mandarin breeding and is important for obtaining seedless fruits. Thus, QTLs responsible for this trait are required to select for it more effectively. Five genomic regions were detected for seed number by using 201 full-sib population, which were crossed reciprocally between Fortune and Chandler . Four QTLs associated with seed numbers were detected in 2014 and 2015. The QTL SN11 was identified on the linkage group Gr9b on locus CTUCH7 with 8.8 %, moreover the other QTLs on the Clementine linkage groups with 8.4, 10.7, and 11.3 % of the explained phenotypic variance, respectively. Yu et. al detected non-repeatable QTLs associated with seed number. These QTLs on scaffolds 3 and 9 of the Clementine reference genome explained phenotypic variance with 21.32% and 19.59 %, respectively .Fruit color is one of the most influential fruit quality attributes of Citrus features because the first thing that affects consumer preference is the appearance and color of the fruit . Citrus peels consist of the pigmented peripheral epicarp or flavedo and albedo .The part responsible for the fruit peel color is the flavedo of the fruit . The color of mandarin peels is usually greenish-yellow, yellow, yellow-orange, orange, and reddish. Fruit pulp color is as important as a fruit quality feature as peel color. Color pigments are responsible for the color of fruit flesh and skin. These pigments are carotenoids and flavonoids. In general, mandarins have a yellow to orange hue. Carotenoids are the primary pigments for yellow to orange colors.There are three main cultivar groups in terms of including carotenoids in Citrus. The first group is the carotenoid-poor group. There are pomelos, lemons and limes, nft vertical farming and grapefruits in this group. The other group is the violaxanthin-abundant group, which includes oranges. The third is the cryptoxanthin-abundant group, primarily containing mandarins . Lycopene, which is responsible for the pink color, is also a carotene and is found in grapefruit such as ‘Star RUBY’ and navel orange such as ‘Cara Cara.’ Anthocyanins are a subgroup of flavonoids that give blue, purple, and red colors to the fruit. Anthocyanins are phenolic compounds responsible for the red color of Citrus fruits such as blood oranges . In addition, with the increase in the popularity of red-colored fruits, mandarin hybrids with red flesh have been introduced to the global market.

These red-fleshed mandarin hybrids are rich in anthocyanins, and examples include ‘Sun Red,’ ‘Early Sicily,’ and ‘Sweet Sicily’ varieties released in Italy . There are some significant genes in the carotenoid biosynthesis pathway in Citrus. They are PSY, PDS, LCYB1, LCYB2, CHY, CHYB, and CCD. The gene responsible for anthocyanin biosynthesis in Citrus is the RUBY gene which encodes an MYB transcription factor. MYB transcription factor regulates anthocyanin biosynthesis with a basic helix-loop-helix transcription factor and a WD40 repeat protein. The expression of the RUBY gene depends on environmental conditions. This gene is upregulated under cold temperatures, causing anthocyanin accumulation in the fruit .Previous QTL studies on fruit color were based on detecting QTLs associated with carotene content in Citrus fruits. Papers reporting QTLs associated with anthocyanin content in Citrus have not yet been published. created a population derived from the female parent ‘Okitsu-46’ and the male parent ‘Nou-5’ . For QTL mapping, EST-based CAPS markers were used to generate linkage maps from 51 progenies and their parents. Their extracts were prepared to measure by HPLC the carotenoid content. According to the results, the A255 map was generated with 345 markers and covered 660cM. In contrast, the G434 map was constructed with 254 markers covering 642 cM. It was cited that there was transgressive segregation for total and each carotenoid contents in progenies. Transgressive segregation arises from the distribution of alleles between parents. It leads to the form of extreme phenotypes in segregated populations compared to the parental phenotypes. QTLs for fruit pericarp and 35 QTLs for pulp were identified. Each QTL for flavonoids in fruit pericarp was explained by phenotypic variance from 30.2 to 71.8%. At the same time, each QTL for flavonoids in fruit pericarp was explained by phenotypic variance from 26.7 to 63.0%. The QTLs for flavonoids were on Chr3, Chr7, and Chr9.Since it is directly related to the external appearance, the rind or peel thickness is a vital fruit quality feature. The peel or rind of the fruit has flavedo and albedo sections and it is reported that mandarins have thinner albedo thickness among other Citrus varieties in general. For example, the albedo thickness of sweet oranges varies from 5 mm to 10 mm. Grapefruits have an albedo thickness of more than 10 mm. Lemons have ±5 mm, and mandarins have an albedo thickness of less than 3 mm on average. Asins et. al performed a QTL analysis associated with rind thickness. A total of QTLs for rind thickness was on the Clementine linkage group, with phenotypic variation explained by each QTL ranging from 9.0 to 21.3% . The juice volume is an essential trait for inner fruit quality. Pulp segments filled with many vehicles are called juice sacs in the Citrus flavedo part. Juice sacs include sugars, organic acids, vitamins, and polyphenolic plant compounds. Juice volume is considered an important criterion, especially for the fruit juice industry . 2 QTLs for juice volume were detected on Clementine linkage groups by Asins et. al . The QTL JV_11 on Cl3 explained 7.4% of phenotypic variance, and JV_11 on Cl4b explained 10.8% of phenotypic variance. In addition, 7 QTLs were identified for juice content . The paper reported that JC was calculated as a percentage of JV and fruit weight . All QTLs related to juice volume and content are defined only on the Clementine map . Yu et. al identified only one QTL associated with juice percentage . JP was calculated by using juice volume and fruit weight. The QTL JP7.2 was on MUR 7.2 with a 2.78 LOD score . In this study, the parents of the mandarin hybrids differ in several fruit traits related to size, seediness, sugar content, and acidity. We sought to achieve a better understanding of the genetic basis of variation in fruit characteristics, including fruit quality and rind color in an outcross F1 population of mandarin hybrids.

Measurements of FDF, berry skin color, and firmness were as described previously. In brief, FDF was determined as the force required to detach the berry from the rachis as measured with a mechanical gauge. A hand-held, temperature compensating digital refractometer and an automatic titrator were used for the following determinations: soluble solids content , pH, titratable acidity . For all these measurements, 10 clusters from each vine were selected and three berries from each cluster were sampled to measure the FDF and three berries for the other measurements. Pre-harvest abscission was determined by counting any abscised berries that had collected in the mesh bags on observation days . Abscised berries were placed in plastic bags and stored in a portable ice box for transport to the laboratory where the integrity of the berry, including the presence/absence of a pedicel, and a wet or dry stem scar was observed with the aid of a binocular microscope at 30× . Berries that abscised pre-harvest and those that fell during harvest, handling or after light shaking constituted the total percentage of dropped berries. The abscised berry percentage was calculated as [/ × 100].Ethephon residues were determined according to the method proposed by Takenaka . For each treatment 30 berries were randomly collected from 10 clusters, stored in a portable FIGURE 1 | Mesh bags to prevent pre-harvest berry loss of Thompson Seedless and Crimson Seedless table grapes. ice box, and carried to the laboratory for analysis. Cartridges SPE NH2 500 mg of Phenomenex activated as suggested by manufacturer were used in the purification step. The purified samples were evaporated to dryness with a rotavapor at 40◦C, taken up with 1 ml of methanol and subjected to derivatization.

One hundred microliters of reconstituted samples were transferred to 1.5 mL eppendorf, diluted with 500 µL of acetone and derivatized by adding 10 µL of trimethylsilyldiazomethane . The reaction vials were maintained at 50◦C for 30 min, then 10 µL of 1 M acetic acid in methanol were added in order to stop the reaction. After centrifugation, 2 µL of the clear upper phase were injected in the GC-MS system.Ethephon application did not affect berry color of Thompson Seedless until 14 days after treatment . At that time, ethephon-treated fruit was darker in color , and had lower C ∗ and a greater h◦ , indicating the fruit were somewhat more yellow colored than non-treated fruit and generally had a more mature appearance. These findings are consistent with other reports that ethephon affects berry skin color by stimulating the accumulation of phenolic compounds . Ethephon treatments clearly reduced FDF because most of the berries on treated clusters were so loosely attached that they abscised before harvest or during handling . However, the few remaining berries on treated clusters were just as tightly held as the berries on non-treated clusters, so no treatment effects on FDF could be measured . A similar result was reported for Thompson Seedless treated with methyl jasmonate, another abscission agent . FDF may decline within a few days of treatment with abscission agents , so timely harvest may be needed when reductions in FDF are large. Abscission agents did not reduce fruit firmness, but FDF and berry firmness decreased from the time of ethephon application whether the clusters were treated or not . As suggested earlier, ethephon at either concentration tested stimulated an almost complete berry abscission from the rachis .

The effects of the two concentrations were similar, with only a few berries still attached to the rachis by harvest time , and the abscised berries generally had dry stem scars . Dry stem scars could be desirable for fresh-cut fruit since the scars help prevent juice leakage and minimize the exposure of interior berry tissues to the atmosphere and to pathogens that might reduce shelf-life or berry quality. However, pre-harvest berry abscission could lead to significant yield losses , though yield loss might be minimized byearlier harvest or the use of catch systems, i.e., nets under the canopy. Ethephon did not affect SSC, pH, or TA . Few studies have examined the effect of abscission agents on grape berry composition, but our results generally agree with Uzquiza et al. who reported few and minor treatment effects on winegrapes. Even though a registered use of ethephon on grape is the promotion of fruit maturity, effects on grape composition are often variable, and ethephon applications to promote fruit maturity are made at veraison, a much earlier stage of fruit development . Abscission agents are applied to mature fruit, so there is less opportunity to affect fruit composition. Moreover, abscission agents quickly initiate the development of an abscission layer between the pedicel and berry . The rapid action of abscission agents necessitates a short time period between application and harvest, further limiting the potential for differences in composition to develop.Ethephon reduced the lightness and purity of the skin color as previously observed for Thompson Seedless, and similarly to that observed by others . The FDF was significantly reduced , whereas SSC and acidity were not affected as in a previous work . A short post-harvest interval limits the possible compositional effects , as discussed above. However, in a previous trial on Crimson Seedless, an increase of tartaric acid, procyanidin P2, terpenoid derivatives, and peonidin-3-glucoside as well as a decrease of catechin and epicatechin was observed after treatments with ethephon a few days before harvest .Treatment with either concentration of ethephon stimulated significant pre-harvest abscission , both >40% and almost 55% at the dose of 2890 mg/L . A similar effect on Crimson Seedless has been recently reported . The treatments tested were less effective at inducing abscission of Crimson Seedless than they were at inducing abscission of Thompson Seedless. Differences among varieties in responsiveness to abscission agents has been previously reported in grape , low round pots and it has also been observed that some table grape varieties are more susceptible than others to “shatter,” or “dry drop,” a post-harvest disorder characterized by the development of an abscission layer between the pedicel and berry . The physiological basis for varietal differences in responsiveness to abscission agents is uncertain, but the application of very high rates of ethephon can induce abscission in varieties that are otherwise non-responsive , suggesting that the less responsive varieties may be less sensitive to ethylene. As observed with Thompson Seedless, SSC, pH, and TA of Crimson Seedless were not affected by abscission agents .The lack of compositional effects are probably due to similar reasons identified and discussed earlier for Thompson Seedless.No present model of solids can predict all microscopic electrical properties of a solid. Two approaches are used in the quantum mechanical study of the electrical properties of solids: free electron models, and bound electron models. Models such as the nearly free electron model, suppose that electrons are free of atoms, and then impose restrictions on how electrons may move. Models such as the tight binding model suppose that electrons are bound to atoms, and then provide ways in which electrons may move.

Meanwhile, bound electron models are necessary for explaining the subtle microscopic properties of solids. A cornerstone the microscopic theory of solids is the Quantum Hall Effect. Within real solids, there is disorder: wave functions and crystal lattices are not uniform or periodic throughout space. With disorder, electrons tend to localize, and materials become insulators. However, within two dimensional materials such as graphene, for certain magnetic fields, even in the presence of disorder, a delocalization of electrons is observed. This conduction is the Quantum Hall Effect, which experimentally exhibits conduction values of exact integer numbers of electrons, or, surprisingly, fractional numbers of electrons known as quasiparticles. Each phase of conduction, or topological phase, is described by a quantum number called the Chern Number, which is an integer that uniquely specifies that topological phase. It is particularly interesting that the transitions between topological phases, known as Plateau Phase Transitions, may described using exactly the same model as for phase transitions between states of matter. This model is the Landau Theory of Phase Transitions, in which a phase transition is quantified through a set of numbers known as critical exponents. Within quantum mechanics, the two basic states of particles are standing waves and traveling waves. Particles in the absence of a global potential take the form of traveling waves and are delocalized. Particles in a global potential take the form of standing waves and are localized. In the global potential created by a magnetic field, electrons behave as standing waves that are circles perpendicular to the magnetic field, also known as cyclotron orbits. In the local, periodic potential created by uniformly spaced atoms, electrons behave as traveling waves of wavelengths such that they will miss the atoms. The Hofstadter Model combines a global magnetic field with a linear combination of atomic orbitals. It is a tight binding model that first assumes that electrons are bound to atoms, and then provides ways to conduct electrons to nearby atoms. Within the Hofstadter Model, the degree of locality is quantified by a distance known as the “localization length.” This length is strongly dependent on the magnetic field and electron concentration, and at Plateau Transitions, this length becomes infinite. In this paper, we proceed by way of an introduction with background given on the theories of electronic conduction, the Classical Hall Effect, Landau Levels, and the Quantum Hall Effect. These sections give a semi-classical and experimental motivation for the notions of localization and topological phase. Next, we introduce the tight-binding model for electronic motion in one dimension, solve it analytically and numerically using the modules, similar results are then obtained for the two-dimensional tight-binding model. The Hofstadter Model is covered, presented as a result of a sum over translation operators, and is then solved numerically using the modules at a variety of fluxes to find the Hofstadter Butterfly. Chern numbers are introduced with the experimental motivation of the Quantum Hall Effect. A method to calculate Chern numbers through the Berry Curvature is presented, as is an efficient algorithm by Fukui, Hatsugai, and Suzuki. This algorithm forms the basis of the Chern number module. This is then applied to a simple model of a Topological Insulator as well as the Hofstadter Model. After that, the concept of localization is introduced and presented in the form of the Anderson Model. This model is then numerically analyzed using modules for transfer matrices and results are described. Finally, the Chalker-Coddington Model is analyzed using the modules and the results are consistent with the literature.Free electron models are a class of models of electron conduction that work well on metals or near-metals. Free electron models assume that there is either no, or very little interaction between an electron and the lattice If there is no interaction with the lattice, then the electron behavior is that of a Fermi gas, where momentum is pF which is given by the Fermi energy _x000F_ F = p 2 F /2m. This is a smooth quadratic function in momentum, and it cannot explain the behavior of insulators which have discontinuities in their energy dispersion.

Other reports have demonstrated an association between L intake or circulating levels and preserving age-related cognitive decline, dutch bucket for tomatoes reducing the risks of certain cancers, coronary heart disease, stroke, metabolic syndrome, and achieving higher levels of physical activity. A systematic review of in vivo, ex vivo, and in vitro studies concluded that L may benefit vascular health by improving endothelial function, reducing inflammation, regulating favorable lipid profiles, and maintaining glucose homeostasis. Systematic reviews summarizing the amount of L needed for cognitive functions and enhancement of gray matter volume estimated that at least 10 mg/d for 12 months could be beneficial. Age must be considered when creating DRI values, since MPOD values are lower in older compared to younger individuals. Whether the proposed intake of L and Z should be based on the amount that can reduce AMD risk, benefit visual maturation in newborns, protect cognitive health, or reduce the risk of other diseases requires further consideration. Sex differences in AMD prevalence must also be considered, especially in relation to pregnancy and lactation, as discussed above. Nevertheless, L and Z are not included for DRI consideration due to inadequate details from food databases, limited large-scale dietary intake studies, and insufficient knowledge regarding their metabolism and biological functions. 

Many continue to advocate for a DRI for L, since it satisfies all nine criteria for bio-active compounds. A rich dietary source of L and Z is goji berry, , also called wolfberry or Gou Qi Zi. The bright orange-red colored oval fruit, has been used for millennia in traditional Chinese medicine for its role in visual health, to provide immunoregulatory, neuro-protective, and anti-inflammatory benefits, and to help regulate liver and kidney meridians . Commercially-available goji berries and their products come primarily from the Ningxia and Xinjiang autonomous regions in western China. Goji berry is known for its high amount of carotenoids, with the Z content higher than any other known food.140,141 In addition to carotenoids, other bioactive compounds found in goji berries include Lycium barbarum polysaccharides , flavonoids, vitamins, minerals, betaine,cerebrosides, phenolic acids, and certain amino acids which may also support the overall health of the eye, particularly when working synergistically. Although the TCM use of goji berry also includes the leaves and bark of the plant, this review will discuss the potential benefits of the fruit on eye health. In addition to a robust amount of Z, goji berries contain modest amounts of β-cryptoxanthin, β- carotene, neoxanthin and L. 

The Z and L content among different varieties of dried goji berries cultivated in Ningxia province ranged from 25 to 152 mg/100g, and 0.3 to 1.9 mg/100g, respectively.143 According to the United States Department of Agriculture food database, one serving of goji berries is 28 g, which would provide up to 42.6 mg of L + Z, depending on the cultivar. Moreover, the predominant form of Z in goji berries is a dipalmitate, found with a diester linkage. The ratio of Z dipalmitate to total carotenoids was up to 55% and 88%, in fresh and dried goji berry fruit, respectively. This esterified form of Z showed a significantly higher intestinal absorption than monoester and free Z due to the high efficacy of hydrolysis, mainly by carboxyl ester lipase. Plasma Z was significantly increased in individuals consuming 15g goji berries daily for 28 days in comparison to those on a habitual diet.  Participants consuming 5 mg of Z dipalmitate extracted from goji berries showed a higher plasma Z concentration than when they consumed the same amount as unesterified Z over a nine to 24 hour period. The high Z content of goji berries has been proposed as a dietary source to reduce the risk of AMD, although studies are limited. In one study, circulating Z levels were significantly higher in healthy older individuals who consumed 10 mg of Z extract from goji berries daily for 90 days. No change in macular pigmentation or soft drusen was observed, but MPOD was not measured. In an uncontrolled trial, individuals with early stage AMD who consume a beverage containing 12 mg of L and 2 mg of Z derived from marigold flower and goji berry, respectively, daily for five months, showed higher circulating levels of L and Z, lower intraocular pressures, and better best-corrected visual acuity scores. Unfortunately, the study lacked a control group, did not test the effect of Z separately, and did not clarify whether the form of Z extracted from goji berry was the dipalmitate. Another study investigating the effects of an herbal formula among healthy adults with dry eyes noted that those chewing tablets containing L , Z , extracts from blackcurrant, chrysanthemum, and goji berry showed dose-dependent reductions in eye fatigue symptoms, improved tear secretion as well as MPOD, compared to placebo. The basis of this formula was derived from TCM, so the multi component formulation could not directly inform the role of any single ingredient. A study in patients with early AMD reported that the MPOD was significantly higher in those consuming 25 g/day of goji berries for 90 days, compared to their baseline levels and to a habitual diet control group. The BCVA was also significantly improved in the goji berry group compared to their baseline values.We recently reported that MPOD and skin carotenoid scores were increased in healthy middle-aged individuals consuming 28 g/day of goji berries five times a day for 90 days compared to a group taking a supplement with 6 mg of L and 4 mg of Z.These results illustrate that MPOD levels can increase in healthy individuals even without early signs of AMD. While these results are encouraging, longer intervention periods with a larger number of participants are necessary. In addition to AMD, goji berries have been studied as a therapy for retinitis pigmentosa, an inherited retinal disease. Patients who consume 0.35 g/d of LBP for 12 months showed a significant improvement in visual acuity and macular thickness, compared to control subjects who did not consume L or Z.156 Examples of human studies that evaluated the effects of supplements containing goji berries on retinal health are shown in Table 1. Based on preclinical evidence, potential benefits of goji berry intake on glaucoma and diabetic retinopathy may also exist. Goji berry extract ameliorated the high glucose-induced blood-retinal barrier disruption in human retinal pigment epithelial cells.Studies reported that LBP showed significant neuroprotective effects over retinal ganglion cells in male C57BL/6N mice and Sprague-Dawley rats with ocular hypertension.In db/db mice, goji berry extract restored the thickness of the retina, the ganglion cell number, blueberry grow pot and the integrity of RPE after daily intake over eight weeks.Although research on the upper limit of goji berry intake is scarce, goji berry allergy risk has been associated with the existence of cross-reactivity to nonspecific lipid transfer proteins from peaches, tomatoes, tobacco, tree nuts, and select pollens.In addition, bleeding symptoms after consuming goji berry juice, tea, or wine have been described in case reports among patients taking warfarin, an anticoagulant medicine.

Although the potential value of foods high in L and Z during pregnancy and lactation has been discussed above, the utilization of goji berry products during these unique periods in a woman’s life needs special caution. Clinical studies of goji berries on eye health have been conducted primarily in Asia, with emerging research reported from Italy, Lithuania, and Switzerland.Potential gene-nutrient interactions must be considered when comparing results from Asian with Caucasian populations.Macular xanthophylls cannot be synthesized de novo in primates. The oxidative defense and blue light filtering characters of L, Z, and meso-Z are important for visual function and potentially reducing the risk of AMD. Because L and Z storage in the maternal body during pregnancy and lactation may become depleted due to active transfer to the offspring, more attention to safe and robust intake of foods and dietary supplements containing these xanthophylls is warranted. Since many infant formula products are not fortified with L or Z, addition of these two xanthophylls should also be considered. The impact of foods and dietary supplements rich in L and Z on MPOD and visual function among AMD patients and in healthy individuals deserves further attention. Since Z may play a different role than L in terms of macular pigment development or protection, better analytical techniques are needed to reassess food composition databases to distinguish these compounds, with greater attention paid to Z as a stand-alone food component, rather than grouping it with other xanthophylls. Goji berries have the highest known content of Z of any commonly consumed food, which also comes in a unique dipalmitate form. Given the increasing rates of AMD worldwide, goji berries, along with L and Z supplements, may help reduce this escalation. As of 2010, safe and accessible drinking water has been explicitly recognized as a human right by the United Nations General Assembly and deemed essential for public health and economic prosperity. Despite this designation, 785 million people lack essential drinking water services globally, and at least two billion people use drinking water sources contaminated with feces. Diseases like cholera, diarrhea, dysentery, hepatitis, typhoid, and polio are linked to such sources of contamination . Despite the risks associated with a lack of essential water services, many drinking water utilities face challenges meeting regulations due to limited water supply, strict budgets, high demands due to population growth, aging infrastructure, and increasingly strict regulations for water quality. Analytical testing and water quality monitoring must be carried out regularly to ensure water is free from priority biological and chemical contamination. Unfortunately, these methods often take multiple days to conduct, in which case contaminated water may already have had negative impacts on public health. These challenges can contribute to public water utilities violating regulations established by the EPA, increasing interest in solutions such as point-of-use filtration, and real-time water quality monitoring. Even if violations are not observed from water utilities there is still potential for contamination between treatment and the tap. This contamination can source back to community water system’s distributional networks and property owner’s premise plumbing . Before reaching taps, consumers can install POU drinking water treatment systems in their water supply lines to provide on-site treatment to the water they consume. These systems encompass many treatment technologies such as membranes, filtration, UV disinfection, activated carbon, etc. The utility of implementing POU filtration shows in its ability to reduce contamination’s acute and chronic health effects. Still, there is little evidence of commercially available technologies to improve the “smartness” of point-of-use water systems in terms of their ability to perform tasks such as monitoring and reporting on water quality Possible ways to enhance the smartness of point-of-use filters include integrating Internet of Things enabled sensor technologies. Commercially available sensors measure parameters including pH, oxidation-reduction potential , and electrical conductivity , which can all be affected by chemical and biological contaminants—making these parameters effective indicators for the overall water quality as well as capable of detecting changes in water quality. When combined with the integrated circuit microcontrollers and micro processing technologies that have emerged in the last decade, there has become a platform for developing robust and customizable data loggers for relatively low costs.

Similarly, another systematic review and meta-analysis included acute and short-term randomized controlled trials concluded that chocolate, cocoa, vertical hydroponic nft system and flavanol intake significantly improved vasodilation function as measured by flow-mediated dilatation , reduced diastolic blood pressure by 1.6 mmHg, and marginally improved the serum cholesterol profile. However, these results were strongest among individuals with moderately elevated blood pressure and untreated hypertension. The vasodilation function in healthy males, measured by FMD, was significantly increased one to four hours after taking a cocoa drink containing 917 mg of cocoa flavanols, compared to baseline values and to those consuming a control beverage with 37 mg of flavanols. The pattern of improvement in FMD from the flavanol-rich cocoa beverage was closely mirrored when participants also consumed 1 or 2 mg/kg of body weight of -epicatechin dissolved in water, suggesting -epicatechin and its metabolites were the main contributors of the vascular effects. Additional research from the same study found that while -epicatechin is a primary contributor to the vasodilation function, dimeric and oligomeric procyanidins that are metabolized by the gut microbiome may also contribute to the vasculo-protection.

In healthy young men, daily intake of a cocoa extract containing 130 mg of -epicatechin and 560 mg of procyanidins for 30 days significantly improved FMD and reduced blood pressure and arterial stiffness as measured by pulse wave velocity , while 20 mg of -epicatechin and 540 mg procyanidins, or a control capsule, did not. However, total cholesterol was decreased after in both groups consuming cocoa, suggesting synergistic effects of -epicatechin and procyanidins, possibly through gut microbiome-mediated catabolism. Cocoa also contains methylxanthines, which are biologically active. While the intake of theobromine and caffeine alone did not result in a significant change in vascular endothelial function measures,interestingly, the combination of methylxanthines plus a high cocoa flavanol drink induced a significant improvement in FMD response than the beverage only. In addition, plasma metabolites of -epicatechin were higher after consuming the flavanol-rich cocoa drink with methylxanthines than when the flavanols were consumed alone, indicating a likely interaction between theobromine, caffeine, and cocoa flavanols on vascular function. Several molecular mechanisms regarding the effects of flavanols on blood pressure and vasodilation have been proposed.

Flavanols such as -epicatechin can increase nitric oxide production directly by increasing endothelial nitric oxide synthase expression. The release of NO consequently increases intracellular cGMP which then induces a relaxation of vascular smooth muscle cells. The increased eNOS activation may also be modulated by flavanols through a calcium/calmodulin pathway by increasing the intracellular calcium concentration, or by phosphatidylinositol 3-kinase/protein kinase B -dependent eNOS phosphorylation. In addition, -epicatechin has been shown to down-regulate the expression of nicotinamide adenine dinucleotide phosphate oxidase by inhibiting the synthesis of vasoconstrictors such as endothelin-1, and therefore increase the utilization of NO. Flavanols may also directly inhibit angiotensin-converting enzyme activity, which increases NO production. Apart from modulating NO production, flavanols may also induce the release of endothelium-derived relaxing factors such as hydrogen peroxide and prostacyclin. Cocoa flavanols also benefit cardiovascular health by inhibiting platelet activation and adhesion. In healthy individuals, platelet aggregation induced by collagen and adenosine diphosphate, and the expression of P-selectin, was significantly decreased compared to a placebo group after the daily intake of 234 mg of cocoa flavanols and procyanidins for 28 days. Some studies have reported that the reduction in platelet aggregation was not different between flavanol-rich darkchocolate or low-flavanol dark chocolate mixed with white chocolate, suggesting potential antiadhesive effects from methylxanthines. While the exact mechanisms to explain the interaction between flavanols and platelets are still under investigation, proposed mechanisms from in vitro and ex vivo models include an inhibition in the expression of endothelial adhesion molecules , and the down-regulation of pro-inflammatory factors such as interleukin -6 and tumor necrosis factor-α, which also decrease the recruitment of other proinflammatory compounds. Mango originated from the Indian subcontinent and has been cultivated for thousands of years. The bark, leaves, roots, and flowers of the tree, and the peel, kernel, and pulp of the fruit, have been used in traditional medicine in tropical and sub-tropical regions throughout the world. The bark and leaves have been used for treating diarrhea and diabetes in Bangladesh, and Ghana, with the pulp and kernel used for hemorrhaging in the lungs and intestines in India. Mango fruit is a rich source of fiber, vitamins C and E, folate, potassium, β-carotene, and phenolic compounds. Dietary intake of vitamins C and E, and β-carotene, are associated with reduced risks for CVD.  Major phenolic compounds reported in mango pulp include mangiferin, quercetin, kaempferol, myricetin, catechin, gallic acid, ferulic acid, protocatechuic acid, and chlorogenic acid.23,57 One study observed that a higher mango intake was associated with improved nutrient intakes, diet quality, and body mass index , factors known to reduce the risk of CVD.58 Clinical trials also suggest that mango fruit may have protective effects against the development of CVD. Daily intake of 200g of fresh-cut Ataulfo mango for 30 daysdecreased blood lipids and increased the plasma antioxidant capacity in healthy adults. In obese men and women aged 20-50 years, supplementation of 10 g/d freeze-dried mango pulp for 12 weeks decreased blood glucose levels but not inflammatory or any cholesterol markers. Another study reported that the daily intake of 400 g of fresh frozen mango pulp significantly decreased systolic blood pressure only in individuals with a BMI of 18-26.2 kg/m2 . In contrast, plasminogen activator inhibitor 1, IL-8, and mitochondrial pyruvate carrier-1 were significantly reduced in individuals when the BMI was partitioned as > 28.9 kg/m2 . In addition, in participants with impaired glucose sensitivity, the supplementation of 100 or 300 mg/d of mango fruit powder with 250 ml water daily for four weeks significantly increased the vasodilation of arteries as measured by the reactive hyperemia index compared to a placebo group. Mangiferin is a unique compound in mango that has been studied for its vasculo protective effects .

A mango bark extract with a high concentration of mangiferin decreased cholesterol in plasma and liver, and reduced oxidative stress in mice. A subsequent human study showed that the daily intake of 900 mg of Vimang® for 90 days reduced a measure of serum oxidative stress compared to a control group among older individuals. In both the animal and human studies, the marker of oxidative stress, nft hydroponic system while considered valid at the time of the study, is now viewed with limitations. In overweight hyperlipidemic individuals, the daily intake of 150 mg of mangiferin for 12 weeks significantly improved lipid profiles and glucose homeostasis. In hyperuricemic rats, mangiferin intake significantly reduced SBP, serum uric acid and inflammatory markers, and increased the expression of eNOS. One potential mechanism to help explain the vasodilatory effect of mangiferin may be due to the increased expression of eNOS, and therefore enhanced the production of NO. Reports have shown that the composition of phenolic compounds in mango varied significantly among different varieties. Mango varieties with high polyphenol content, such as Ataulfo, may play a more prominent role in cardiovascular health, but the interaction between the polyphenols, carotenoids and other bioactive compounds in mango must be considered. Further studies may also focus on the potential effects of mango by-products on metabolic health, since the total concentration of phenolic compounds is higher in the kernel, peel, leaves, and bark compared to the edible fruit. Such explorations may be useful in processing what is considered as agricultural waste into useful extracts.Low-bush blueberry and high-bush blueberry are two common species originally grown in North America. Native Americans have a long folklore history of using both types of blueberry plants to treat rheumatism and infection.  Anthocyanins are responsible for the red, blue, and purple color in ripe berries. Blueberries are one of the most abundant sources of anthocyanins in commonly consumed fruits. The total anthocyanin level in fresh blueberries is significant, reaching up to 487 mg/100g. Blueberries also contain appreciable amounts of proanthocyanidins and hydroxycinnamic acids , along with vitamins and minerals, fiber, and small quantities of flavonols and flavanols. Epidemiological studies suggest that a higher dietary anthocyanin intake is associated with a lower risk of hypertension, and reduced arterial stiffness in women, though these studies do not specify blueberries as the sole source of these bioactives. A meta-analysis of 19 cohort studies reported that the dietary intake of anthocyanins was associated with a decreased risk of coronary heart disease and CVD mortality, but not myocardial infarction, stroke, or total CVD risk. The effects of blueberries on markers of CVD risk have been studied. In obese postmenopausal women with pre- and stage I-hypertension, daily consumption of 22 g of freeze-dried blueberry powder containing 469 mg of anthocyanins for eight weeks significantly reduced systolic and diastolic blood pressure by 7 mmHg and 5 mmHg, respectively, and arterial stiffness measured by brachial-ankle PWV, compared to their baseline values or to a placebo group. In healthy males, FMD was significantly increased one, two, and six hours after the intake of 34, 57, and 80 g of blueberry powder mixed in water , compared to a control drink. However, no changes were seen in arterial stiffness measures. In addition, the increase in polyphenol metabolites and decrease in neutrophil NADPH oxidase in plasma were correlated to FMD, suggesting that the phenolic metabolites after blueberry powder consumption effectively improved vasodilation functions by elevating the bio-availability of NO through inhibition of NADPH oxidase. Later the research group identified that the FMD improvements were mainly due to anthocyanin metabolites. The blood pressures of overweight and obese smokers who consumed 250 g of blueberries for three weeks showed no significant changes from baseline values . Among mid-aged women who were at risk for type II diabetes, daily consumption of 240 ml of wild blueberry juice with 314 mg of anthocyanins forseven days significantly improved serum nitrates and nitrites, but no change were noted for in glucose metabolism parameters, cholesterols, inflammatory markers, platelet adhesion molecules, vasodilation, or blood pressure, compared to baseline and the placebo group. Taken together, the above results suggest that clinical trials with blueberries may need require a few weeks or longer of regular intake in order to observe clinically significant changes. Similar to blueberries, the American-cranberry is also a plant that is native to North America and has a long history of botanical uses by indigenous people, such as for urinary tract disorders and diarrhea. Cranberries are rich in numerous phenolic compounds, including A-type procyanidins, anthocyanins, flavanols, benzoic acid, and ursolic acid. Due to the extremely low sugar and high tart and astringency nature, cranberry products often contain added sugar or are blended with other fruits to improve palatability. To date, no systematic reviews or meta-analysis has noted an efficacy of cranberries in reducing CVD risks. However, potential cardio-protective effects may exist due to mechanisms discussed above.Goji berry , also termed wolfberry or Gou Chi Zi, has been used in TCM for its “eye-brightening” effects for millennia. In Chinese culture, goji berries can be consumed as a snack, an ingredient in soup, or as a tea alternative. Goji berries have the highest known content of Z of any commonly consumed food. In addition, goji berries also contain modest amounts of β-cryptoxanthin, β-carotene, neoxanthin and L.

For blackberries, costs for a mature crop are shown for the second through fifth production years, and total $43,406 per acre per year. Harvest costs again represent the vast majority of total costs, at 81% and 71% of total costs for raspberries and blackberries, respectively. For raspberries, cultural costs represented a much smaller share of total costs at $4,656 per acre, roughly half of which was for trellis and tunnel management. Blackberry cultural costs totaled $5,709 per acre, of which over half was for pruning and training canes.Each study also includes an analysis of potential net returns to growers above operating, cash and total costs for a range of yields and prices. When evaluating net returns above total costs, gains are shown for higher yield and price points; losses are also documented at many lower yields and prices . Farms with productive soils, experienced managers, optimal production conditions and robust market plans generally realize higher net returns. In contrast, farms with less-than-optimal production conditions, reduced yields, poor fruit quality or inexperienced managers may contribute to lower net returns. Results from the strawberry analyses show that on a per acre basis, organic strawberries tend to be more profitable than conventional berries, even with lower yields.

Organic price premiums explain the result; in this example price per tray for organic strawberries ranged from $12 to $18, while price per tray for conventional berries ranged from $7.30 to $11.30. Prices for second year conventional strawberries were slightly lower still to account for a portion of the crop that was diverted to the freezer market. Net returns for both caneberries were mostly positive. Other noteworthy entries in all recent berry studies include per acre costs for pest control advisers , management of invasive pests and food safety and regulatory programs for water and air quality. Though each alone represents a relatively small portion of total costs, they provide readers with insights into the changing nature of berry production activities and costs over time.Cultural practices in the berry industry have evolved to address changes in soil, water and pest management needs. Based on historical trends, and to meet both industry needs and consumer demands, we expect to see new varieties continually developed over time. Businesses have responded to consumer and market demands for fresh, safe and organic products by implementing food safety programs and/or transitioning more lands to organic production.

Water and air quality programs have been developed to comply with state regulatory requirements. In the past, growers customarily hired those with expertise in financial and market management; they now also enlist the support of experts in food safety, organic agriculture and environmental quality to assist with farm management. But challenges remain, and management of key agricultural risks — including those for production, finances, marketing, legal and human resources — have become increasingly important. Invasive pests pose significant management and regulatory constraints and increase production, financial and market risks. Two recent examples are light brown apple moth and spotted wing drosophila . LBAM infestations can lead to loss of part or all of the crop because of field closure from regulatory actions, increasing production and financial risk. SWD presents substantial market risk to growers in that its larvae can infest fruit and render the crop unsaleable. Growers minimize the risk of loss from these two organisms with the routine use of PCAs. PCAs monitor fields more frequently than growers alone would be able to do, identify pests and recommend actions, for example, the use of pheromone mating disruption for LBAM and field sanitation for SWD. Since their introduction, the soil fumigants CP and MB have unquestionably contributed to the expansion of the berry industry. However, the full phaseout of MB as a pest management tool — it will no longer be available for use in berry production after 2016 — presents both production and financial risks. While a substantial research commitment has been made to finding alternatives to MB, nothing has yet come close to offering the same level of protection from the large-scale loss to soil pathogens or the gains in productivity associated with the application of CP and MB as synergistic preplant fumigants. We anticipate that the berry industry will adapt to the MB phaseout by using alternative fumigants and preplant soil treatments, but these are likely to carry a higher level of risk for berry production in the short term and may lead to a decrease in planted acreage and production. However, this may also stimulate an even more robust research agenda directed towards soilborne diseases and plant health to minimize disruption to the industry. Reliance on fumigants as the primary strategy for pest management is almost certainly a thing of the past. Instead, adoption of integrated approaches, including alternatives to fumigants, to manage diseases, weeds and other pests will be key to sustaining berry production over the longer term . Labor is also a current and significant challenge for growers of berry crops. Social and demographic changes in Mexico — the source of a majority of the area’s agricultural labor — have resulted in markedly lower immigration rates into the United States, a shrinking labor pool and upward competition and wage pressures for the agricultural workers who remain . In recent years, growers have reported difficulty in securing and retaining sufficient numbers of workers to ensure timely and effective farm operations. The lower production figures seen in strawberries in 2014 may in part have been the result of an insufficient labor pool from which to draw . However, no known regional employment or wage data are available to specifically document this. Some growers minimize labor risk by paying higher wages and providing year-round employment when possible. However, these strategies can be difficult for some businesses to justify economically. Arguably, the area’s berry industry, and agriculture more generally, stacking pots increasingly face political risk. Immigration legislation that may assist with the current labor challenge languishes at the federal level, with major policy changes unlikely before 2017 . Farming practices are under ever more scrutiny by consumers, local municipalities and state and federal agencies. Soil fumigants and pesticide use have been the focus of many intense debates and discussions, especially in Santa Cruz and Monterey counties. At the time of this writing, several new regulations related to pesticide application notifications, pesticide and fumigant application buffer zones and worker safety have been proposed by the California Department of Pesticide Regulation or the U.S.

Environmental Protection Agency but have not yet been finalized. It is anticipated that implementation will begin in 2017, with full compliance required in 2018. And, as California struggles through a fifth year of drought, water use, quality and cost has become a more robust part of the local, state and federal discourse, with directives issued and new legislation proposed. Compliance with each new directive or regulation presents production and logistical challenges for growers and can be costly to manage. Although it is unlikely that regulatory pressures will lessen in the future, there is every expectation that growers will continue to adjust business practices to meet or exceed any new requirements or standards. The economic sustainability of individual farming operations and the area’s berry industry in total will ultimately be impacted by and continue to evolve with the ever changing business environment, and by an array of risks and challenges.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 non-climacteric 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, 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?

For instance, Giambelluca et al. found more rapid warming at elevations above 800m in Hawaii, and a review by Pepin et al. found enhanced warming with elevation in high mountain regions globally . Therefore, higher elevation sites in our study may also be experiencing more warming. We do not have temperature records for our six sites so we cannot separate temperature trends or anomalies from other elevational impacts. Nonetheless, the lack of a consistent elevation effect could be due not only to the confounding effects of other topographic features , but also to lower rainfall and increased warming in some higher elevation sites. From the early studies of Whittaker ecologists have long recognized the influence of aspect on plant composition and vegetation dynamics in montane environments. Yet rarely have investigators tied performance or mortality of individuals within a species population to subtle differences in aspect that occur within a single population . Here we found that aspect was the strongest predictor of dieback over time, with plants in SW aspects showing greater dieback than plants on more north-facing aspects. This influence of aspect may be the result of direct negative effects of sun exposure and higher temperatures ina semi-arid environment or maybe indirect whereby influences of aspect are mediated through aspect-effects on soil and microbes. For example, Gilliam et al. 2015 demonstrated greater soil organic matter accumulation and different microbial community composition in N facing compared to S facing slopes. Regardless of mechanism, the finding that SW aspect is detrimental to these plants could be important for restoration practitioners seeking to identify promising sites on the landscape where restoration of individual species may be more successful .

Another source of variability both within the landscape region we studied, and across Southern California chaparral, which was not directly measured in this study, is the occurrence of fog. The Santa Barbara region experiences predictable fog in May and June, and late summer cloud shading and fog events have been demonstrated to reduce vapor pressure deficit , slow plant seasonal water loss and provide direct foliar uptake of water . We observed significant variation in fog intensity between sites, particularly at night during the early summer months , even between sites on a given night. Sites C and D in particular often experienced heavy fog drip, while other sites remained dry. We found plants saturated by fog to exhibit higher water potentials on such nights, similar to findings in Mahall et al. , suggesting that fog drip may be sufficient enough to percolate to shallow roots. As mentioned previously, A. glauca is considered to be a relatively shallowly rooted chaparral shrub species and has been shown to be sensitive to even small changes in rainfall . Additionally, some coastal California shrub species have been shown to exhibit foliar water uptake from fog , though this has not yet been demonstrated for A. glauca. Regardless, variations in summer fog patterns have previously been shown to influence late-season water potentials in other Arctostaphylos spp. , possibly causing populations with greater exposure to fog to have higher “safety margins” with which to avoid cavitation . Similarly, Emery et al. have shown that fog can reduce the rate of water loss in coastal shrublands in California. Mortality of A. glauca during drought documented in other studies occurred in more interior sites, with less fog influence.

Thus, we believe the role of local and regional variation in fog occurrence in A. glauca drought resistance warrants further research.While dieback severity increased over time, the fact that no new plant-level mortality was observed during the study supports previous findings that A. glauca are resilient to prolonged drought. The relatively more mesic climate of Santa Barbara may have contributed to resiliency in our study, compared to studies in more xeric shrubland regions that reported high mortality of A. glauca during the same drought . Landscape heterogeneity, while likely confounding some of our results, may also be advantageous in creating differential water availability across the landscape, thus providing refuges that can ultimately allow resilience of A. glauca populations in a region. However, severe and widespread dieback, like that which we observed, still represents a threat to healthy ecosystem functioning, and has implications for fuel management in regions that are already fire-prone. Lower elevations and exposed, southwesterly slopes may continue to seethe highest levels of dieback during future drought events, and thus should be identified as high-risk and potential focus areas for management.Supported metal catalysts consisting of finely dispersed metal nanoparticles on high-surface-area supports are key in realizing cleaner and more efficient chemical conversions by lowering the energy cost and increasing the selectivity to the desired product. Efficient use of precious metals is facilitated by dispersing the catalytically active metal in sub-10 nm parti-cles. Preventing the growth of these small NPs into larger agglomerates, for example, at elevated temperatures and pressures, is challenging and remains a major cause of catalyst deactivation. Traditionally, high surface-area supports are used to retard NP sintering by increasing the interparticle spacing. Most prominent approaches are to distribute the particles in well-defined mesoporous supports, to encapsulate the individual NPs in porous oxides, to induce a thin metal oxide coating via strong metal-support interactions, or by incorporating the NPs in a microporous framework, such as, for example, zeolites. These designs drastically increase the thermal stability of the NPs and successfully protect the particles from sintering and Oswald ripening during catalysis. However, the encapsulation can lead to a partial loss of catalytic activity due to blockage of the NP surface by the surrounding metal oxide. A key challenge is to mitigate this effect, hydroponic nft and to develop methodologies to probe the chemical accessibility of the NPs in these novel catalyst designs. An emerging class of materials developed by our group that addresses this challenge is raspberry-colloid-templated catalysts . The RCT materials exhibit exceptional thermal and catalytic stability, while maintaining high catalytic activity and selectivity. The material design consists of an ordered macroporous metal oxide framework with sub 10-nm metal NPs distributed at the pore walls. The colloidal preparation approach that involves infiltration of the metal oxide precursor into the assembled sacrificial colloids decorated with catalytic NPs offers high versatility of the structural design, that is, particle size, metal composition, metal oxide composition, and micro- and macro-porosity can be tuned independently. The robust, macroporous matrix allows facile mass transport throughout the catalyst. So far, the potential of the RCTs for thermal catalysis has been demonstrated in selective alkyne hydrogenation, HD exchange, CO oxidation, oxidative alcohol coupling, and the oxidation of volatile organic compounds. A unique feature of the RCT catalysts is their excellent stability during catalysis and thermal treatment, allowing long catalyst lifetimes and facile reactivation via thermal treatment. A particularly noteworthy finding by Shirman et al. is the observation that not only the stability but also the activity of the RCT catalysts can be strongly enhanced compared to commercial catalysts, allowing drastic reductions in precious metal use and in energy cost in oxidation catalysis.

The origin of the superior thermal stability of the RCT catalyst has remained an open question thus far. Furthermore, the crystal structure and accessibility of the catalytic NPs, in particular at their interface with the support, are still unknown despite the relevance of these properties to understanding the origin of sinter-resistance of these materials and their activity in catalysis. Here, by using theoretical modeling, 3D electron microscopy, and epitaxial overgrowth, we assess the NP embedding, the crystal structure, and the chemical accessibility of the metal NPs in RCT catalysts and discuss the implications of these structural characteristics for the catalysts’ functional properties, and in particular for NP stability.The RCT preparation relies on multi-step synthetic route . The first step comprised the preparation of the metal NPs and amidine-functionalized polystyrene colloids . Here, the metal particles consisted of 96 atom % Au and 4 atom % of Pd, abbreviated as Au96Pd4, and were capped with polyvinylpyrrolidone . However, the desired composition, size, and shape of both the NPs as well as the size and functional surface groups of the PS colloids can be readily altered, independent of the subsequent synthesis steps, allowing a tailored design of catalysts for specific chemical reactions. Next, the metal NPs were attached to the PS colloids, yielding so-called raspberry colloids . An ordered colloidal crystal of raspberry colloids assembled by solvent evaporation was infiltrated with a pre-hydrolyzed silica sol-gel solution . In the final step, the PS template and stabilizing ligands were removed via calcination , generating an ordered macroporous silica support with metal NPs decorating the pore walls. Additionally, the high-temperature calcination ensured the mixing of Pd and Au into a homogeneous alloy. The resulting RCT catalyst contained ≈5 wt.% bimetallic Au96Pd4 NPs as determined with inductively coupled plasma mass spectrometry . The weight loading closely matches the theoretical weight loading of 4.8 wt.% calculated based on the synthesis parameters, indicating that no metal leaching occurred during the RCT preparation. The sinter-resistance of the NPs in the RCT catalyst is evident from the overlapping size distributions before and after thermal treatment at 800 °C in static air . The average particle sizes before and after treatment closely match and are 7.5 ± 2.5 and 7.4 ± 2.4 nm, respectively. Additional long-term stability experiments show that thermally treating the catalyst for 10 h in either oxidizing or reducing gas atmospheres does not lead to particle growth . These findings are in line with previous reports in which particle growth was not pronounced in RCT catalysts at temperatures up to 950 °C, and in strong contrast to significant agglomeration and growth of NP in silica-supported catalysts in which the NPs were introduced after the macroporous silica framework was prepared. Thermal treatment of the latter already led to particle growth to > 20 nm after thermal treatment at 500 °C in static air. In addition to the thermal stability of the NPs during synthesis and in oxidizing and reducing atmospheres, the long-term stability and sinter resistance of the PdAu and Pd NP RCT catalysts have been demonstrated for a range of reactions under catalytic conditions: CO oxidation, methanol oxidation, 1-hexyne hydrogenation, and oxidation of simulated exhaust mixtures. It has been shown that the sinter resistance is maintained in RCT catalysts even at high metal loadings and small NP sizes. We postulated that the infiltration step in the synthesis procedure is crucial in determining the NP–silica interface and the degree of NP embedding into the matrix after calcination. More specifically, the geometry of the NP pocket within the silica support is defined by the wetting of the surfaces of the gold NPs and PS colloids by the infiltrating silica sol-gel solution.