Nature Ecrogrow

Similarly, in the outer model, changes in MVs reflect a change in their LV, and thus connect the outer model with MVs to the inner model of LVs. For instance, the MV PC3 has a negative correlation with the LV leaf shape , so that as the value of PC3 decreases, it reflects as a corresponding increase in LV leaf shape . This change is represented as an increase in the roundness of the leaf. This then corresponds to a positive change in yield , which is in turn a reflection of fruit biomass . The model indicates that photosynthesis has a strong positive influence on both fruit BRIX and vegetative biomass but has a negative impact on fruit yield. As photosynthetic rates increase , fruit BRIX increases, but at the sacrifice of yield, an inverse relationship which has long been known . Leaf shape has a negative relationship with vegetative biomass, which corresponds to the decreased leaf complexity with the Potato Leaf Morph . However, leaf shape has a strong positive influence on both fruit BRIX and yield , suggesting that leaf shape influences fruit quality as seen previously by Chitwood et al. . The effect of leaf shape on fruit quality does not work through leaf sugar, as this correlation was not significant. Our leaf sugar measurements were completed in the glasshouse, owing to the complexity of the chemical analyses required, growing raspberries in container and as such the model was tested without leaf sugar. No significant causative relationship changes occurred in the model upon omitting the leaf sugar values.

While our work does not implicitly study mechanisms, the negative relationship between leaf sugar and fruit BRIX is of interest, and may provide some avenues for future research into the mechanisms underlying impact of leaf shapes on fruit quality in tomato. Fig. 6 displays the effect of each trait on the overall output of the plants . Leaf shape has no strong contribution to vegetative biomass. Although shape shows a negative relationship with biomass, this influence is minimal when compared with photosynthesis . However, leaf shape shows the largest influence on both yield and fruit BRIX, with photosynthesis second, and is the only positive contributor to yield . This positive correlation is from rounder, Potato Leaf Morph-like leaves, while narrower leaves have the opposite effect based on the PC contributions to leaf shape. The negative effect of photosynthesis on tomato fruit yield and the strong contribution of leaf shape to yield and BRIX are novel findings that run counter to the interpretation of fruit quality improvement, as increased photo assimilate should result in more available sucrose to stronger sinks such as fruit . To test the model performance we used PLSPREDICT on the entire heirloom dataset used to build the structural model. Table S8 shows the mean absolute percentage error and Q 2 value for the complete model. We also used part of the dataset that included ABC Potato Leaf and Aunt Ginny’s Purple in a similar analysis . The complete model has c. 20–30% error for each LV, which is expected given the diversity of genotypes in the dataset, with fruit weight giving the highest MAPE, at 93.2% . The Q 2 value for most variables is positive and shows that they have relevance in the predictive performance, with the exception of leaf sugar, which is slightly negative .

In the case of ABC Potato Leaf and Aunt Ginny’s Purple, two lines selected randomly to test the model on individual cultivars, a significant increase in Q 2 and decrease in MAPE is seen for all LVs except leaf sugar . This indicates that the model is substantially stronger in predictive performance for individual cultivars, but also predicts well with the complete model. To evaluate the predictive performance of our model on additional datasets, we used data from two other cultivars grown in the same field, M82 and ‘Lukullus’, that were not used to construct the model. PLSPREDICT was used in SMARTPLS 3.0, along with the structural model constructed using the heirloom cultivars, to test the model performance by use of training sets and hold out samples, both taken from the M82/’Lukullus’ dataset. By using the leaf shape PC values, we were able to compare the predicted mean values for the remaining MVs, or the predicted measured values, against the actual measured values and evaluate the relative performance of the model. Tables 2 and 3 show the results for M82 and ‘Lukullus’, respectively. PC values for leaf shape are not included as they are input variables and used for predicting the other values. For M82 the predicted median values compared with the actual median values showed under 1% difference for all except leaf complexity, which had a percentage difference of 8.42% . This indicates that the model was under predicting the leaf complexity of M82 by c. 8%. ‘Lukullus’-predicted values were also under 1% different, except for leaf complexity and stomatal conductance which varied by 2.56% and 1.31%, respectively .

In addition to the predicted values PLSPREDICT also tests the model performance and reports the root mean square error, mean absolute error, and MAPE for each of the MVs tested . The MAPE shows the accuracy of the predictions, with lower percentages representing better performance. Leaf complexity for both cultivars showed the largest MAPE values, 201.2% and 26.5% in M82 and ‘Lukullus’, respectively . The M82 MAPE indicates that the model does not predict leaf complexity well for mid-level complexities such as 18 but does improve at high-end leaf complexities near 40 . Most heirloom cultivars had low leaf complexities , potentially explaining the poor performance in predicting leaf complexity for M82. Contrary to previous findings , we found that leaf complexity does not impact yield or BRIX, and only impacts vegetative biomass, so this inaccuracy would only impact vegetative output predictions by the model. ‘Lukullus’ has indeterminate growth like the heirlooms analyzed here, but M82 is determinate; however, the predictive accuracy of the model was still good, indicating its usefulness in assessing field performance of other tomato cultivars.The primary focus of crop improvement has been on fruit traits and photosynthesis , with some studies focusing on how sugars are moved from source to sink. Despite heirloom varieties with the Potato Leaf Morph being prized for fruit quality by the gardening community, vegetative traits such as leaf shape have been relatively ignored in breeding efforts. In this study we investigated the role of leaf shape on fruit quality by measuring both input traits and output traits for 18 heirloom cultivars. All these cultivars were classified as Potato Leaf, but varied greatly in their leaf shapes, development, and fruit quality . We found that these lines do not vary significantly in overall photosynthetic capacity, or their usage of light when available , suggesting that the variation in BY among these cultivars was not a result of improved/decreased photosynthetic capacity. While our measurements for photosynthesis do not show significant difference when PAR is available, the PARi differed between cultivars based on their growth patterns . All cultivars exceeded 1200 lmols m2 s 1 of PARi but varied in the later weeks between 1200 and 2000 lmols m2 s 1 . Combining multiple complex physiological and morphological measurements into informative relationships has proven difficult and has limited our understanding of how these different traits impact each other . Focusing on any one part, such as photosynthesis or fruit sink strength, raspberry container size while providing improvements , occurs at the expense of a comprehensive understanding of the overall relationships between these traits. Analyzing the individual PCs revealed significant differences in leaf shape among the heirloom cultivars, with several having stronger Potato Leaf Morphs and higher BY values , with some correlation between these traits. Potential epidermal shape changes that could arise from leaf shape changes and that could influence yield would relate to stomatal number. A previous study in 2002 analyzed several tomato cultivars developmentally and histologically and found no real differences between these cultivars. This and another study in 2010 suggest that there are no gross anatomical differences between these tomato cultivars. We used PLS-PM to combine all these measured traits, using the modeled final harvest data as input to find causative relationships . Strong relationships among gas exchange, light, and photosynthesis were expected, along with a strong positive effect of photosynthesis on vegetative biomass .

Photosynthesis has a strong positive effect on fruit BRIX, both directly and indirectly . Increased photosynthesis results in lowered leaf sugar content, and a concomitant increase in fruit BRIX. It is possible that increased sugar production from photosynthesis results in higher rates of transport of sugars out of the leaves and into sinks. The mechanisms that regulate source–sink relations and sugar distribution are still not fully understood on a whole-plant physiological level ; however, based on our model, increased photosynthesis negatively impacts total yield . While photosynthesis does lead to increased sugar production and is shown in our model to drive higher sugar content within existing fruit, it does not provide a means to increase yield. Leaf shape, specifically rounder, less lobed leaves, has a positive effect on both fruit BRIX and yield . Of all the factors measured here, only leaf shape positively influenced yield, with other paths having negative influences . Rounder leaves still drive slightly increased photosynthesis indicated by the thin arrow , which results in increased fruit BRIX. This path should also result in decreased yield. However, leaf shape has a strong positive and direct correlation with yield that overcomes the negative impact of photosynthesis and leads to increased yield as well as BRIX . Conversely, with narrow leaflets there is a small negative impact on photosynthesis which should result in increased yield, but narrow leaves have a direct negative impact on yield which is stronger than the photosynthetic pathway . The strong causative relationship among leaf shape, fruit BRIX, and yield suggests that leaf shape impacts both high fruit BRIX and increased number of fruits, probably by modulating sugar distribution, therefore bypassing the direct impacts of photosynthesis itself . How leaf shape affects this distribution is unclear, as it does not act directly through leaf sugar content, or through strong regulation of photosynthesis to improve yield . A recent study looked at the diversity of leaf shape in sweet potato . Any correlations between leaf shape and yield traits in this species would be of interest and help to establish general principles. The whole-genome phylogenetic analysis of 23 tomato cultivars showed many of the Potato Leaf Morph cultivars were closely related to each other, with the exception of Brandywine, though it did not show the origin of the C-locus mutation . To address this and identify if this morphology was selected for in breeding, we performed PHYLONETWORKS analysis . This analysis showed several hybridizations between Potato Leaf Morph and nonPotato Leaf Morph cultivars, and probably a unique incidence of the C-locus mutation in Prudens Purple . PHYLONETWORKS analysis of chromosome 1, 6, and 12 specific common SNPs each showed unique hybridization events, distinct from those seen in the WGS analysis . The PHYLONETWORKS analysis suggests multiple hybridization events with Potato Leaf Morph-containing cultivars. Potato leaf cultivars have been suggested to increase disease resistance compared with regular leaf varieties and may have been selected for this reason or for other asyet-unknown benefits present. We have shown that leaf shape strongly impacts the overall fruit quality in tomato, with rounder, less lobed leaves giving rise to higher yield and higher fruit BRIX. Photosynthesis, surprisingly, has a negative impact on yield while still positively contributing to fruit BRIX. Using data from cultivars not included in making our path model, we also showed that the model has a strong predictive performance for linking leaf shape to BY and could be used to potentially predict the outputs of a cultivar using leaf shape data . Our work shows the importance of leaf shape to yield and BRIX across a wide array of genetic backgrounds, implicating leaf morphology in playing a significant and previously unidentified role in tomato fruit quality.Eighteen heirloom tomato varieties identified as having a range of fruit types, including cherry and beefsteak tomatoes, and several intermediate types, were analyzed. These tomato varieties also differed in fruit production timing from early to late, and the type of leaf morphology.

The At2S3:RUBY results demonstrate that RUBY could be an effective marker for Arabidopsis transformation. Furthermore, betalain was not widely transported from the sites of synthesis to other tissues as we did not see any red color in leaves . We also expressed RUBY under the control of the Arabidopsis YUC4 promoter . YUC4, which encodes a key enzyme in auxin biosynthesis, was shown to express in small regions of embryos, leaves, and flowers. GUS signals were observed in leaf tips and apical region of a gynoecium in YUC4 promoter:GUS transgenic plants. We observed similar patterns of betalain production in YUC4:RUBY lines .The maturation of citrus fruit is coupled with significant changes both in peel color and in pulp sugar and acid content. The process is believed to be triggered and regulated by external and internal stimuli . Known plant hormones involved are ethylene, abscisic acid and gibberellins . Ethylene does not trigger an autocatalytic ethylene production and a corresponding respiration peak in the non-climacteric citrus fruits as it does in apple, raspberry container growing banana and other climacteric fruits. Yet, ethylene does trigger color changes in citrus fruit peel . Reduced ethylene production was found in some late-ripening mutants along with lower expression of ethylene biosynthesis genes, such as ACC synthase and ACC oxidase .

Ethylene involvement in citrus fruit maturation is also manifested by its regulatory effects on the expression of carotenoid biosynthetic genes, chlorophyllase genes and other fruit ripening– related genes . Increase in the ABA content was found to be concomitant with color development and carotenoid accumulation in peels of maturing citrus fruit . Upregulation of ABA biosynthesis and signaling genes were observed during citrus fruit maturation . Exogenous ABA treatment accelerated fruit coloration in Ponkan, whereas treatment with nordihydroguaiaretic acid, a synthetic ABA inhibitor, retarded fruit coloration and juice acid degradation . An ABA-deficient sweet orange [Citrus sinensis Osbeck] mutant, Pinalate, displayed slowed fruit degreening . The content of ABA and pigments was lower in the flavedo of a stay-green mutant of Ougan in comparison with that in the motherwood Ougan . Comparative transcriptional and proteomic analyses between late-ripening sweet orange mutants and their corresponding wild types also showed an essential role of ABA in citrus fruit maturation . In contrast to the promotive effects of ethylene and ABA, GA treatment delays the color break of citrus fruits. Applications of GA to the fruit of an early maturity pomelo-grapefruit hybrid could effectively retain the fruit color and prevent postharvest fruit senescence . Pre- or postharvest treatment of fruits with gibberellic acid retarded pigment changes in Satsuma mandarin . Auxins have been suggested to be important for the attainment of ability to ripen and for the coordination in the subsequent steps of the ripening process of both climacteric and non-climacteric fruits .

The content of indole-3-acetic acid , the primary natural auxin, was particularly high during fruit set and started to decline before the onset of fruit ripening . Application of exogenous auxins delayed the fruit ripening process in strawberry , tomato , banana and grape . However, an initial increase in IAA content is necessary for stimulating ethylene production and fruit maturation in climacteric fruits such as apple , plum , peach and pear . The IAA also regulates carotenoid biosynthesis by repressing the expression of phytoene synthase , ζ -carotene isomerase, phytoene desaturase and carotenoid isomerase, and promoting the expression of β-cyclase 1 and β-carotene hydroxylase during tomato fruit ripening . Impairment in IAA biosynthesis and signaling could also result in abnormal fruit ripening . A recent report showed that the exogenous application of NAA accelerated chlorophyll degradation and carotenoid accumulation in Satsuma mandarin fruit treated with two color-retarding agents, GA and prohydrojasmon . Extending fruit maturation will extend its marketable season and hence increase the value of the fruit. Shatangju is very popular among citrus consumers in China for its superior fruit quality. The main drawback of the cultivar is its short fruit marketing period. Previously, we identified a mutant of Shatangju mandarin , designated as ‘Yuenongwanju’ . Fruit maturation of the MT was significantly delayed by ∼50–60 days compared with that of the WT. Here, by comparing the transcriptomes and the contents of multiple metabolites and two phytohormones of the MT and WT fruits, we provide insight into the role of IAA in citrus fruit maturation.

Consistent with the expression trend of the DEGs involved in the metabolism of ABA and IAA, a gradual increase in the ABA level and a steady decrease in the IAA level were observed in immature fruits of both genotypes. As shown in Figure 6A, an ABA peak was observed at the color change stage in both the MT and the WT fruits. There was no significant difference between the ABA peaks of the two genotypes. However, the ABA contents were unexceptionally lower in all MT fruits at 180, 210, 230 and 250 DPA than in the WT fruits of the same ages. Figure 6A also showed the changes in fruit endogenous free IAA content in both genotypes. It was clear that the fruit free IAA content gradually decreased in both genotypes as the fruit size increased. The IAA content stabilized at a lower level, ∼0.6 ng g−1 FW, after the fruits reached full size. Comparatively, the MT fruits contained significantly higher free IAA than did the WT fruits at all time points prior to 250 DPA. The higher free IAA content in the MT fruit prompted us to investigate whether IAA was responsible for the delayed maturation of the MT fruits. The MG WT fruits, ∼210 days postanthesis, were therefore picked and treated in vitro with IAA in 2019 and 2020 . As expected, the content of free IAA in fruits treated with IAA was significantly higher than that in water-treated and untreated fruits . Treatments with 100 and 1000 μM of IAA increased the pulp IAA levels by ∼15- and 25-fold, respectively, within 24 h. The IAA concentrations in fruits of both treatments decreased rapidly but were still significantly higher than those in the controls after 48 h . It was not unexpected that the IAA-treated WT fruits exhibited a significant delay of color change . Both the untreated CK and the ddH2O-treated fruits completely turned yellow after 25 days of storage at RT. Meanwhile, the fruits under 100 μM IAA treatment had just started changing color, and those under the 1000 μM IAA treatment were still deep green . At 60 days post treatment, fruits treated with 100 μM IAA had almost completely changed color, but those treated with 1000 μM IAA had only slightly changed color .Fruit ripening is controlled by different hormones. For example, blueberry plant pot the ripening of climacteric fruits such as tomato, peach, banana and papaya is regulated by ethylene and IAA . Ripening of non-climacteric fruits, such as strawberry, grape and sweet cherry, is predominantly regulated by ABA and auxin . Auxin, unlike its positive role in enhancing the biosynthesis of ethylene in climacteric fruits , works as a negative regulator, inhibiting ABA biosynthesis in non-climacteric fruits . Citrus fruits are classified as non-climacteric fruits. Their ripening process is therefore considered to be mainly controlled by ABA, although accumulating evidence has suggested that ethylene plays some role, at least in chlorophyll degradation in fruit peel . The role of IAA in citrus fruit ripening has not been extensively explored. In this study, a late ripening mutant of Shatangju mandarin whose fruits mature in February, almost 2 months later than common Shatangju, was characterized. It was found that the younger fruit contained more free IAA and less ABA than did the older fruit, suggesting that IAA might act as a counter maturation agent . Furthermore, exogenous IAA treatment significantly delayed color change in WT fruits . Additionally, significant differences in either IAA or ABA levels existed between the MT and the WT fruits of the same age. That is, the IAA content was higher, whereas the ABA content was lower, in the MT fruit than in the WT fruit. These differences between the two genotypes no longer existed only in fruits that were experiencing color change. Taken together, it seemed that a higher level of free IAA and a lower level of ABA were necessary for maintaining the growth of the citrus fruit until color change. Intriguingly, a match in the IAA or ABA content could always be found between an older MT fruit and a younger WT fruit.

In other words, the MT fruits were able to reach the same physiological status of the WT fruits later , indicating that a slower growth in the early stages of the MT fruit development should be responsible for the lateripening phenotype. Contrary to our findings, a synthetic auxin, NAA, was found to be able to accelerate chlorophyll degradation and carotenoid accumulation in GA- and PDJ-treated Satsuma mandarin fruit . Nevertheless, studies have shown that NAA had bipolar effects on citrus, as manifested by the fact that it either prevents fruitlet drop if used at lower concentrations or promotes fruitlet drop if used at higher concentrations . The fruit of the late-maturing mutant exhibited a significant delay in CB and a dramatic change in the expression of numerous genes. The most pronounced internal changes were that the IAA level was elevated, whereas the ABA level was lowered in growing fruit by the mutation. Correspondingly, some of the IAA and ABA metabolic genes were differentially regulated. Endogenous free IAA levels were determined collectively by the genes of IAA biosynthesis, conjugation and degradation pathways . The indole-3-pyruvate pathway was considered to be the predominant pathway for IAA biosynthesis in higher plants . The rate-limiting step of the pathway that catalyzes the conversion of IPA to IAA is controlled by the YUCCA gene family , and three members of the family, CrYUCCA3, CrYUCCA5 and CrYUCCA8, were found to be expressed differentially between WT and MT . Although the expression of CrYUCCA3 was opposite to that of CrYUCCA8, the three genes combinedly showed a decrease in their expression, which was in agreement with the trend in changes in free auxin. An auxin degradation gene, DAO1, was abundantly, but differentially, expressed between WT and MT, and remarkably, its expression was twice as high in MT as in WT at 230 DPA . It seemed that the expression of a GH3 gene was also elevated in MT fruits . Logically, the elevation in the expression of IAA degradation and conjugation genes should lead to a reduction in the IAA levels in MT fruits, but our findings were the opposite. Multiple studies have shown that alterations in the auxin levels could be corrected by the compensatory changes in auxin metabolism since auxin homeostasis is vital for maintaining normal cell function . For example, Arabidopsis plants lacking AtDAO1 activity did not result in an increase in free IAA levels since the conjugation of IAA was concomitantly elevated . For ABA biosynthesis, the rate-limiting step that cleaves xanthoxin to produce 9-cisviolaxanthin and 9-cis-neoxanthin is controlled by the NCED gene family, and two members of the family, CrNCED3 and CrNCED5, were found to be expressed differentially between WT and MT . Remarkably, the expression level of CrNCED5 in the MT fruit was only half that of the WT fruit at the last two physiological stages, the CB stage and the CC stage . The NCED5 gene was considered to be a key regulator in ABA biosynthesis in citrus fruit . The gene’s expression was closely corelated to the content of ABA in the two studied genotypes . Fruit ripening is a complex biological and physiological process involving changes in many primary and secondary metabolic processes . One of the pronounced changes is the fruit color. Citrus fruit’s color development is correlated with a gradual reduction in green color and a steady increase in mainly orange and yellow colors . Metabolic changes in chlorophylls and carotenoids therefore play a very important role in the coloration of citrus fruit peel . Our RNA-seq data showed that the genes related to chlorophyll and carotenoid metabolisms were differentially expressed. Three genes, CrPSY2, CrZDS1 and CrLCYb2a, which encode three key enzymes in the carotenoid biosynthesis pathway, expressed at higher levels in WT fruit than in MT fruit at 230 and 250 DPA .

S1-bZIPs play essential roles in plant growth and development, especially seed maturation, root growth, and flower development . For example, the transcript abundance of AtbZIP53 is markedly induced during the late stages of seed development . AtbZIP53 enhances the gene expression associated with seed maturation by specific heterodimerization with group C-bZIPs . AtbZIP11 and AtbZIP44 play a role in embryogenesis. AtbZIP44 shows high transcript levels at the early stage of seed development and is involved in micropylar endosperm loosening and seed coat rupture via its interaction with the promoter of AtMAN7 . The atbzip44 knock-out mutant shows slower germination and reduced expression of AtMAN7 . In Populus, the binding of poplar bZIP53 to the promoter of IAA4-1 and IAA4-2 inhibits adventitious root development . In horticultural plants, three S1-bZIP members are highly expressed in grape seed , but their regulatory mechanisms have yet to be elucidated. For example, CsbZIP-06 is highly expressed in female cucumber flowers and ovaries . Transgenic lines over expressing mORF of BZI-4 show reduced flower size and impaired pollen development . Over expressing AtbZIP1, AtbZIP53, tbz17, MusabZIP53, and FvbZIP11 shortened internode length, and stunted vegetative growth . FabZIPs1.1 and FvbZIP11 have been shown to be involved in fruit ripening in strawberry . Banana MabZIP91 and MabZIP104, large pots plastic which belong to S1-bZIP subgroup, showed high transcript abundance during fruit development and ripening .

These studies illustrate the various roles of S1-bZIPs as a regulator of plant growth and development .The S1-bZIP subgroup, with their functional diversity in all plants, reflects their importance as regulators. The literature covered in this review suggests that the small but unique and crucial S1-bZIP transcription factors play essential roles in the balance of carbon and amino acid metabolism, plant growth and development, and stress responses . S1-bZIPs also play important roles in regulating fruit quality and stress response. Through heterodimerization with group C-bZIPs, S1- bZIPs orchestrate an array of downstream transcriptional and metabolic control. However the C group bZIP dimerization partners of many S1-bZIPs have yet to be identified. The S1- bZIPs regulate sugar signaling and amino acid metabolism under energy-deprived conditions, which involves the Sucrose Induced Repression of Translation mechanism of the uORFs and through interaction with the SnRK1 pathway. However, further research is needed to explore whether and how SnRK1 and TOR kinase interact with C- and S1-bZIPs complex. The SC-uORF negatively regulates the translation of S1-bZIP mORFs and, in turn, downstream targets of the S1-bZIPs, which further affect fruit quality and other metabolite biosynthesis. Evidence suggests that over expression of S1-bZIP mORFs significantly increased the fruit sugar content and sweetness, showing the potential for improvement of fruit quality .

In addition, functional diversity and specificity among the S1-bZIPs need to be further defined. Using substitution of conserved amino acid residues in the DNA-binding domain could be a useful approach to clarify specific interconnections among S1-bZIPs and their dimerization partners in horticultural plants . Using CRISPR technology to create indel mutations in uORF start codons or enhancing the expression of S1-bZIPs using fruit specific promoters could provide broad applications to control the levels of sucrose and other nutrients for the improvement of the quality of fruits, vegetables, and flowers, and to improve stress response without the detrimental effects on plant growth and development in horticultural plants .A thorough investigation of primate diets, and how primates alter their diets in response to variation in food availability, is fundamental for understanding primate behavior, ecology and morphology. Periods of resource scarcity may have particularly important impacts on primate fitness because during these times feeding competition can be intense and food quality poo. While such periods can have disproportionate impacts on primate feeding adaptations and sociality, they occur infrequently in some environments. Long-term observations of primate feeding behavior and concurrent assessment of plant food availability are therefore necessary to sample across the full range of variation within the diet and to encompass periods of high and low resource availability. The need for long-term data sets is particularly acute in Southeast Asia because most forest types there exhibit dramatic, supra-annual fluctuations in fruit production that exceed the magnitude of variation in food availability characteristic of other tropical forests.

Mast fruiting events are periods of super-abundance of resources, and are characteristically followed by periods of extreme food scarcity. These phenological cycles are linked to EL Niño Southern Oscillation events and consequently occur at irregular intervals that are unpredictable from the perspective of vertebrate frugivores. Due to the hyper-variability in food availability in the Dipterocarp forests of SE Asia, dietary changes in response to food availability can be dramatic, with some primate species incurring negative energy balance during periods of low resource availability. Studying the responses of frugivores to these fluctuations in food availability—especially the responses of multiple taxa that differ in their dietary adaptations, life histories, and feeding strategies–can shed light on the evolution of primate feeding adaptations. A useful way to understand dietary responses to fluctuations in food availability is to categorize dietary items based on their use and availability, and in particular to distinguish between preferred and fallback foods . Preferred foods are generally high-quality foods that are easy to process and are eaten more often than would be predicted based on their availability. Foods that are consumed more during periods when preferred foods are scarce are termed fallback foods. Comparative studies of primate diets are particularly informative for understanding how responses to resource availability drive evolutionary processes. For example, the African grey-cheeked mangabey has a relatively high degree of dietary overlap with the sympatric red-tail guenon . L. albigena possesses much harder tooth enamel than C. ascanius, some of the hardest tooth enamel found in extant primates. The foods that L. albigena consumes during times of resource scarcity are thicker and harder to process than foods eaten by C. ascanius, and the difference in tooth enamel thickness between the two species can be explained by the foods they consume when resources are scarce. Comparative studies can also be useful for understanding how resource availability influences primate population biology. For example, the population density of white-bearded gibbons is limited by the availability of their fallback foods, whereas red leaf monkey population density is limited by the availability of high quality, preferred foods; these differences may be due to differences in the life histories of the two species. Gibbons and leaf monkeys provide an excellent comparison for investigating the effects of resource variability on primate ecology because they are similar in body size, but have different social systems, life histories and diets. Gibbons generally live in male-female pairs and have relatively slow life histories , whereas leaf monkeys live in single-male, multi-female groups and have relatively fast life histories. Gibbons and leaf monkeys are classified as frugivores and folivores/gramnivores, respectively. Gibbons are generally considered ripe-fruit specialists and possess few morphological adaptations to process low-quality foods, whereas leaf monkeys, like all colobine monkeys, have morphological adaptations such as complex, multi-chambered stomachs, thin tooth enamel and high shearing cusps that facilitate the consumption of leaves. In this study, we conduct a dietary analysis of two sympatric primate species, red leaf monkeys , square planter pots and white-bearded gibbons in Gunung Palung National Park, Indonesia using plant phenology data and primate feeding observations collected over 66 months. We examine the feeding ecology of sympatric populations of gibbons and leaf monkeys to: 1) characterize and compare gibbon and leaf monkey diets, identify the genera consumed and their importance, the relative contribution of different plant parts to overall diets, and overall dietary richness, diversity and overlap; 2) analyze feeding selectivity for each primate species; and 3) assess how these primates respond to temporal variation in fruit availability. Specifically, we make the following predictions: compared to gibbons, leaf monkeys will have higher dietary richness and diversity; prefer more genera, and avoid fewer genera; and show shifts in types of plant parts consumed in response to variation in overall fruit availability. We make these predictions based on evidence that leaf monkeys have morphological and physiological adaptations to process a wider variety of foods than gibbons.We conducted this study at the Cabang Panti Research Station in Gunung Palung National Park, West Kalimantan, Indonesia from September 2007 through February 2013. At CPRS, mean gibbon group sizes are 4.32 individuals and mean home range size is 43 ha ; mean leaf monkey group sizes are 5.77 individuals and with 90 ha mean home range size.

There are seven floristically distinct forest types at Gunung Palung National Park, but for the present analyses we focused on the five forest types that exhibit mast fruiting as the non-masting forest types have dramatically different phenological patterns and plant species composition. We operationally define mast fruiting events as periods where there was at least a three-fold increase in fruiting stems above the mean proportion of stems fruiting in all other months. We recorded daily maximum and minimum temperature and rainfall at the field station at CPRS .Each month, AJM, field managers, or trained Indonesian field assistants walked two replicate census routes in each of the seven forest types found at CPRS and collected data on gibbon and leaf monkey feeding behavior. Inter-observer reliability was ensured through extensive training, periodic checks of distance measures, and regular quizzes to assess the accuracy of plant and vertebrate species identifications. Observers were randomly rotated across habitat types and census routes, and average encounter rates and detection distances are highly concordant between observers. Standard line-transect methods allowed for the collection of statistically independent feeding observations and avoided the potential for pseudo-replication that may occur when multiple feeding observations are collected from the same group on the same day. We systematically walked fourteen, spatially segregated line transects, at a consistent speed between 0530 and 1200 hrs . For any group or individual encountered while feeding, we recorded the first item consumed by the first individual seen. We collected feeding data on all age and sex classes, thus adults and juveniles of both sexes were included in our analyses. Because data were collected across multiple forest types and many groups, the results reflect the diet for the population, rather than potentially idiosyncratic observations of a single group. Following collection of feeding data, observations along the vertebrate census route continued so that multiple feeding observations were not made from the same group on the same day. We collected additional feeding data during targeted focal observations of gibbons and leaf monkeys. We selected target groups at random from among the known groups at the site . After contacting the target primate group , we randomly selected a focal individual of any age-sex class and followed until it began feeding. Data collection on focal follows continued for 30 minutes, at which point a new focal individual was randomly chosen. We did not record a new feeding observation from the focal animal until it had travelled to a different tree or liana to ensure that multiple feeding observations were not recorded from the same individual plant. We collected the following data for each primate group encountered on transect routes and during focal follows. For the plant fed upon by the first primate individual sighted, we recorded the identification of the plant eaten , location , size , and growth form of the plant; the part being eaten ; the maturity stage, if applicable ; the number of animals feeding; and an estimate of the total crop size. We gathered one feeding observation every 3.6 days, on average . In previous analyses, we found there were no significant differences in the use of plant genera collected during line transect surveys or focal follows, therefore we lumped feeding observations together to increase sample size.To assess spatial and temporal variation in food availability, we monitored the reproductive behavior of tree and liana stems located in fifty 0.1 or 0.2 ha botanical plots . Each month all stems in every plot were carefully examined with binoculars and assigned to one of six reproductive states . Determination of fruit ripeness stages was based on changes in size, color, and texture, using categories developed over the last 30 years for each plant taxon. Mature fruits are full-sized fruits that are unripe but have seeds that are fully developed and hardened; ripe fruits are the final development stage prior to fruit fall, usually signaled by a change in color or softness.

Following Rain 1, the relative abundance of 44 OTUs changed significantly on the tomato fruit surface, including 10 members of the Enterobacteriaceae and 3 members of the Xanthomonadaceae, all of which increased in average relative abundance. Tere were no significant differences at the family level between 9/9 and 9/13, however there were significant differences in 6 low-abundance bacterial families between 9/9 and 9/25. For example, relative abundance of the family Rhodobacteriaceae increased steadily over the study period , from ~0.35% to 3.5%. Te most abundant taxa tended to fuctuate throughout the study period irrespective of proximity to rainfall. On average, the Pseudomonadaceae increased in relative abundance from 9/9 to 9/13 , decreased on 9/17 , only to increase again after Rain 2 and decrease again by 9/25 . Te increase from 9/9 to 9/13 was not statistically supported, however the increase from 9/17 to 9/22 was statistically significant . Relative abundance of the Oxalobacteriaceae also increased significantly following Rain 2, from 0.5% to 2.9% , following a small borderline insignificant increase after Rain 1, from 9/9 to 9/13 . This temporary post-rainfall increase in relative abundance of the Oxalobacteriaceae mirrored results seen on cucumber.On tomato leaf surfaces, increases in α-diversity around rainfall were not discernible, plastic gutter with an average of 150 OTUs per sample. Tough not significant, the pre-rain timepoint had the highest median number of OTUs per sample .

A core microbiome of 27 OTUs was observed in all leaf samples. Twenty-one OTUs common to all pre-rain samples were not observed 1 day post-rain, and only 6 new OTU introductions common to all 1 day post-rain samples were detected. No OTUs were found to be common across all samples between 1 day and 4 days post-rain timepoints other than the core 27 common to all 3 dates, while 7 OTUs shared among all 4 days post-rain and pre-rain samples were recovered. On the other hand, shifs in β-diversity were detected across the full sampling period; timepoint significantly influenced bacterial community structure at a rarefaction level of 8,200 sequences per sample when analyzed with unweighted UniFrac distance and Bray-Curtis dissimilarity . However, weighted UniFrac analysis did not reveal a significant effect . No taxa shifted in relative abundance between the pre-rain and 1 day post-rain timepoints, but changes in the average relative abundance of some low-abundance families were detected between the pre-rain timepoint and 4 days post Rain 2. Te Unweighted UniFrac distances between averaged sample groups from each timepoint suggested a weak but consistent shift in community structure over the course of the sampling period, but also considerable variation within samples from the last timepoint .Cucumber fruit surfaces yielded a higher number of bacterial OTUs, 281 , compared to tomato fruit surfaces, 232 . Similarly, bacterial community structure differed significantly between the 2 fruit types .

Several of the most dominant taxa differed in average relative abundance between cucumber and tomato fruit surfaces, including Pseudomonadaceae , Xanthomonadaceae , Methylobacteriaceae , and Microbacteriaceae . Enterococcaceae were higher in average relative abundance on cucumber compared to tomato fruit . Taxa of the Rhizobiaceae family were prevalent on both tomato and cucumber fruit surfaces and were not significantly different . Similarly, members of the Enterobacteriaceae were dominant on both tomato and cucumber fruit surfaces . .In order to more directly address the food safety implications of potential bacterial community changes in response to rainfall, samples were screened for presumptive generic E. coli, frequently used as an indicator of fecal contamination. In total, tomato leaf samples had significantly higher levels of presumptive E. coli compared to tomato and cucumber fruit samples . No significant differences were observed between sampling dates for any sample type, although presumptive E. coli counts and variability in the data increased in all samples following Rain 1 and the highest individual values within each sample type were observed on 9/13, the day following the first rain event .Although the impact of precipitation on fruit and vegetable crop microbiomes has not been directly investigated, it has long been understood that increased plant disease and food safety risks can succeed rain events by enhanced dissemination of pathogens, splash or fooding and by provision of more favourable growth conditions. Shifts in precipitation and periods of drought could also affect bio-control microorganisms, altering the suppressive potential of microbiomes. Reaching a more nuanced understanding of the precise effect of precipitation on crop microbiomes and the microbial dynamics that ensue is valuable to the application of systems thinking and approaches to crop protection, especially when confronted with climate change and increasing severity and duration of rainfall and drought periods. Te consistent seasonal fuctuations recently described in the airborne microbiome captured in rain and snow imply that bacterial introductions via rain could be predictable during sequential rain events in a given agricultural region, hence allowing for crop management decisions to be made with regards to anticipated microbial dynamics in response to drought or precipitation.

In this study, assessment of microbiomes associated with cucumber and tomato surfaces using high quality amplicons of the 16 S rRNA gene provided a novel contribution to our understanding of the impact of rainfall on epiphytic bacterial communities at the time of crop harvest. This work demonstrated an increase in bacterial species diversity on cucumber and tomato fruit surfaces following rain events, at times accompanied by shifts in bacterial community structure. Several new bacterial taxa were introduced to the cucumber and tomato carpoplanes following rainfall and persisted at low abundance in the days following precipitation. This points to a window of time following precipitation during which introduced taxa may become newly established in the phyllosphere, potentially constituting a period of high risk for plant disease and food safety. On cucumber fruit surfaces, the relative abundance of several of the most dominant taxa changed following rainfall, often fully or partially returning to pre-rain proportions within 4 days. Furthermore, overall bacterial community structure on cucumber fruit shifed significantly in response to rain as measured both when incorporating phylogenetic relatedness of bacteria present and including the relative abundance and phylogenetic relatedness of bacterial OTUs . Tomato fruit-associated bacterial communities shifted throughout the study period when assessed by OTU richness and the unweighted UniFrac metric. By contrast, this observed sustained increase in diversity on the tomato fruit surface was not clearly accompanied by shifts in measures that take relative abundance into account, being only detected using Bray-Curtis dissimilarity but not weighted UniFrac. An additive effect considering multiple rain events could explain these differences, however other drivers cannot be ruled out. Phyllosphere microbiome responses to rain have not been investigated in crop systems but reports from rhizobacterial communities suggest that below ground microbiota may be more resilient. Studies on the effect of precipitation on the rhizosphere microbiome appear to reveal that rain has a limited impact on bacterial community composition but can drive changes in relative abundance of taxa. Our study indicated that phyllosphere bacterial assemblages of cucumber and tomato are responsive to precipitation events. This is turn suggests thatphyllosphere microbiomes could be amenable to modulation with the aim of achieving desired outcomes such as disease resistance, enhanced food safety and stress tolerance. Unlike fruit surface communities, blueberry container tomato leaf surface community α-diversity remained largely consistent across all sampling dates; a decrease in the number of OTUs observed 1 day post-rain was not statistically supported. A shift in community structure was observed in the phylloplane over the course of the sampling period, however, in concordance with results from tomato fruit, the incorporation of abundance data into the UniFrac metric diluted the effect. For both tomato fruit and leaves, this indicates that any changes in community structure could likely be attributed to shifts in low-abundance taxa. In tomato fruit, these changes in low-abundance taxa could be observed both through shifs in relative abundance and introduced taxa.

The diminished effect on leaves, on which little post-rain seeding of novel taxa was detected, could be the result of the strong influence of the plant host on species recruitment. However, abiotic conditions on the leaf could also be a factor, with higher relative humidity surrounding leaves as a result of transpiration and trapped moisture within the layer of abundant trichomes. By contrast, mature tomato fruit lack stomata and trichomes, such that moisture from rain could have significantly changed growth conditions by increasing the amount of free water available to microorganisms. Compared to results seen on tomato, microbiota associated with the cucumber carpoplane were more responsive to weather-related changes, although only one rain event was evaluated in the case of cucumber. Fruit and vegetable crops harbor distinct bacterial communities that could be based on inherent differences in plant surface topography and nutritional profile. Differences in cropping practices could also partly explain differences between the two fruit crops. Cucumbers were grown on plastic culture on the ground, while tomatoes were staked upright. Cucumber fruit lying directly on plastic mulch were lef both more exposed to direct rainwater contact and closer to the soil, increasing the potential for splash. Newly introduced taxa may have originated from rain or transferred via splash or wind from soil or nearby plant parts. While the sampling dates were selected to surround rain events, other weather dynamics during the sampling period could not be controlled for and likely influenced plant-associated microbiomes as well. Differences in barometric pressure and wind speed or reduced UV stress due to cloud cover could have influenced crop phytobiome dynamics or interacted with the factor of rain. Furthermore, rainfall may have been correlated with larger scale ecosystem changes. For example, insect visitation, which can affect plant microbiomes may have been limited during the rain event but elevated in the days following precipitation. Pesticides were applied to tomato plots during the sampling period, on the evenings of 9/9 and 9/16. It is possible that these applications could have influenced microbiome structure and diversity, however phyllosphere bacterial communities tend to be fairly robust in the face of pesticide application. Pesticides were not applied to the cucumber plot and similar but more discernable responses to rainfall were detected on cucumber fruit. Prior to Rain 1, the region experienced a long drought with negligible rainfall since the previous major rain event 3 weeks before the study began. The increase in diversity observed following Rain, 1 but not Rain 2, could be explained by drought-induced suppression of bacterial diversity at the start of sampling, not replicated prior to Rain 2, which occurred only 9 days later. Due to the close proximity of the rain events, it is possible that bioaerosols were less prevalent during the second rain. Plants release microbes into the atmosphere preferentially on sunny, dry days, and there were few of those between the two rain events. Unfortunately, cucumber data for Rain 2 were not collected due to seasonality and a lack of availability of high-quality fruit samples, such that we cannot address whether the difference between Rain 1 and Rain 2 is mirrored on cucumber. Te shifts in bacterial OTU richness and in some cases community structure that we did observe following rain events could have been the result of direct inoculation by rainwater-associated microbiota or by other factors associated with rainfall. Rain could physically remove microbes from the plant surface, opening up a niche for others to fill. Alternatively, increased moisture and relative humidity in the air before, during and after rain events could favor rapid growth of certain taxa at the expense of others. In previous work we noted that E. coli levels on lettuce spiked after moderate precipitation but plummeted after heavy rainfall, suggesting that the effect of rain is a balance between new species introductions, stimulating growth conditions as a result of enhanced moisture, and a depleting effect, depending on rainfall depths. In this study, we saw an increase in variation of E. coli on fruit and leaves, but no significant hike in population levels, suggesting that this taxon is not an adequate indicator of bacterial community shifts in the phyllosphere. While it is important to understand the local influence of isolated rainfall events on microbial dynamics in agriculture, in the future it will also be important to consider the influence of weather patterns on a larger scale. In addition to the direct impact of rainfall on phytobiomes, prolonged wet or dry periods could influence plant health and immune responses, and storms could lead to wounding, creating opportunities for pathogens to infltrate plant tissues.

We used a full-factorial experimental design to test for the effects of pollen limitation on fruit production and foliage variables of whole trees experiencing four resource treatments: normal water and nutrients; reduced water/normal nutrients; no nutrients/normal water; and reduced water and no nutrients. In each of these resource input combinations, we applied three pollination treatments: supplemental hand pollination to maximise cross-pollination; open-pollination with flowers exposed to bees freely foraging in the field; and pollinator exclusion, accomplished by caging trees during flowering. The 12 treatment combinations were randomly assigned to individual trees and replicated five times in adjacent rows .Hand-pollination was carried out from 20 to 28 February using Padre pollen that had been harvested before bud opening and stored at 20 °C to maintain viability. Prior to application, pollen was thawed and used immediately to ensure viability. We hand-pollinated all open flowers using small brushes every 2–3 days until about 90% of all buds had opened. The last 5–10% of flowers that opened late in the blooming season were frequently characterised by deformed or missing female or male parts. For the pollinator exclusion treatment, we covered individual trees from shortly before blooming started in February to the end of bloom in early March with 1.5 m² 9 2-m tall cages constructed of aluminium tubing and cloth with a mesh size of 0.8–1.0 mm.

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.

The phenology of the novel host, such as the timing of flowering and fruiting, also affects the ability of a tephritid to use a new host . Importantly, host chemicals are key drivers when herbivores encounter a novel host and serve as attractants and barriers to adaptation . Phytochemicals include volatile compounds and secondary metabolites that serve as attractants or defensive compounds to herbivores, such as tephritids. Volatile compounds allow tephritid adults to select among potential hosts while in fight, similar to fruit color. Once tephritid flies overcome the volatile chemicals of a potential new host, they eventually make contact with the host fruit, and then they must adapt to any secondary metabolites present to successfully colonize the host fruit. These chemical and nonchemical cues of a potential novel host fruit act as selective pressures on tephritids when a novel host is encountered . These selective pressures involve visual identifcation; behavioral selection; and physical, chemical, and neurophysiological responses by tephritid flies to the novel host fruit . There is likely a genetic basis for each of these processes, which suggests that various genes are involved in regulating the host plant expansion of tephritids. Therefore, black plastic plant pots increasing our knowledge of the categories and roles of these genes in regulating host expansion will deepen our understanding and allow for improved management strategies for tephritid fruit flies.

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 activities of vacuolar and cell-wall invertases were not reported, and it might therefore be assumed that most of the cell-to-cell movement is through the symplastic pathway.Photo assimilates were detectable in the stalk of the juice sacs as early as 6 h after 14CO2 feeding, as found by pulse-chase experiment. However, with continuous exposure, the kinetics of radioactivity accumulation were higher between 24 and 48 h of exposure . Sugar-metabolizing enzymes were not monitored in the stalk separately from the juice sac, but the same mechanisms are likely to be operating in both parts of the juice sacs.As the edible part of the fruit, sugar metabolism and transport in the juice sac have received more attention than in other fruit parts. Photo assimilate transport proceeds to the inner part of the juice sac . Following 1 h of 14CO2 feeding to a source leaf next to grapefruit fruit, and 1 week of translocation, about 60% of the label was found in the juice sacs, with similar results in Satsuma mandarin . A maximal rate of radiolabel accumulation in pulse-chase experiments was reached between 24 and 48 h of labeling . Movement from the stalk to the distal part of the juice vesicle is relatively slow, and may take up to 96 h in the case of pomelo juice vesicles, which can reach 3 cm in length . Interestingly, drainage planter pot whereas in grapefruit juice sacs, most of the labeled assimilates were recovered as sucrose, in Satsuma mandarin, fructose was predominant .

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 effectiveness of the selected bacterial carrier to protect encapsulated phytochemical compounds was assessed based on the thermal treatment of both the encapsulated phytochemicals and the phytochemicals in a control juice matrix. In summary, this study demonstrates the potential of using inactivated probiotic bacterial cell carrier for the binding and encapsulation of phytochemicals from a complex juice matrix and characterizes the binding and encapsulation efficiency of diverse phytochemicals and stability of encapsulated compounds in bacterial carriers using a combination of chemical analysis, spectral imaging, and antioxidant properties.Fruit juice contain a variety of phytochemicals, which in general can be classified into alkaloids, carotenoids, nitrogen-containing compounds, organosulfur compounds, and phenolics. Many in vitro and in vivo studies support that the antioxidant property of the phytochemicals plays a major role in their essential health benefits such as anti-inflammation and anti-carcinogen. To characterize the overall efficiency of encapsulating complex profiles of phytochemicals using the bacteria cell carriers, the relative encapsulation efficiency was measured based on the difference in antioxidant concentration of the juice sample before and after incubation with cells. To quantify this ratio, drainage pot antioxidant concentration in juice sample and juice residue after the encapsulation process was measured using the FRAP assay.

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.

Genetic map building with quantitative trait loci analysis is needed to identify possible candidate genes and to predict molecular markers associated with fruit quality characteristics. The most preferred molecular markers have been used as single nucleotide polymorphisms , polymorphic insertions or deletions , or microsatellites recently. Genome-wide association studies and linkage mapping need high-throughput molecular marker assays. Although genetic mapping studies are still limited in mandarin due to the insufficiency of phenotype data and the complexity of fruit quality traits, some studies have been conducted on fruit quality traits that are important for Citrus breeding . The QTL analysis would help select parents and improve hybrids of the parents with the target gene of a favorable trait by preventing the long juvenile period of Citrus and eliminating Citrus breeding expenses . Improvements in next generation sequencing and genotyping array technologies have helped to understand the genetic basis of quantitative trait variation. SNP genotyping became the most widely used genotyping method for GWAS and QTL mapping due to being inexpensive and producing many, greenhouse vertical farming codominant SNPs. In addition, SNP genotyping can be performed with SNP arrays or produced by genotyping-by-sequencing , or whole-genome sequencing.

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.

Grapevines were cane pruned with 12–14 nodes per cane. Vines were drip irrigated from May to September . A single irrigation pipeline was positioned on the soil with three drippers for each vine . Soil water potential was kept below −300 kPa, and leaf water potentials were maintained at values < −0.6 MPa. Fertilizer addition, pest control, and other vineyard operations were conducted according to local practices. A randomized block design was used with three blocks and three treatments, and each treatment in the block consisted of six grapevines selected with a uniform number of clusters. Each treatment consisted of: control, ethephon at 1445 mg/L, ethephon at 2890 mg/L. The concentrations used in this trial were established on results obtained in preliminary studies . Ethephon was dispersed in water with 0.1% of a surfactant and applied directly to the clusters of vines selected for abscission treatments. Clusters from control vines were treated with water containing the surfactant only. The ethephon or control solutions were applied with a handheld sprayer until run-off when the fruits reached sufficient soluble solids for harvest . After the berries dried, raspberry cultivation pot each cluster was enclosed in a mesh bag to collect any berries that may abscise.Berries were sampled before treatment, 2 h after treatment and in successive days, as reported in Table 1.

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.