Month: December 2024

The opposite is true for Rr and no effect is found for Rs. Overall CAwr has the highest relative fecundity with the highest fruit production . Average number of fruit damaged for each lineage correlates: strongly with Rs and Rr fruit production, Rs and Rr potential reproduction and Rs and CAwr final plant weight, and moderately with CAwr fruit production, potential reproduction and Rr final plant weight. In all the cases the covariance values are positive. No correlation was found with days to germination . Debris due to granivory – Debris material due to granivory was only found under plants in unprotected plots. In general the debris due to granivory was a mix of viable and unviable seeds as well as fruit wall segments. Some seeds were found bared, with no fruit material around them, while others were either fully or partially covered by fruit wall segments. The largest amount of debris due to granivory was found under CAwr plants in unprotected plots as well as the largest number of potentially viable seeds. Most of the fruit segments were found broken and empty. The average values are listed in table 2.6. Although the debris due to granivory data was based on a small sample size, I calculated the average proportion of seeds removed for each plant by granivory in two different ways. One of the calculations was done as described in the methods. The other calculation included the subtraction of the potential “viable” seeds found in the debris due to granivory and averaged for each lineage . Subsequently, I compared both averages. In the case of both progenitors there are minimal differences. However, procona system for the hybrid derived-lineage there is a 10% reduction of seeds removed.

Granivore behavior – We observed and filmed house finches actively manipulating and feeding on fully formed Raphanus fruits. No other avian species were observed on Raphanus plants although there are at least five other granivores regularly occurring in this location . At the different plots, the birds visited the plants daily in an almost predictable fashion. In all cases I observed males, easily recognizable for their characteristic red coloration around the face and head. The peak of house finch foraging activity corresponded to the period in which the fruits were fully formed but still green. We never observed the house finches feeding out of the debris on the ground; they appear to only choose fruits attached to the plants.The house finch Carpodacus mexicanus, an avian granivore, affected the reproductive patterns of three Raphanus progenitors and hybrid lineages in dissimilar ways. When exposed to the granivore, the hybrid-derived CAwr had proportionally more fruits damaged relative to its progenitors . Overall relative fecundity of CAwr was significantly reduced compared to protected plants of the same lineage . When exposed to the granivore, CAwr had lower fecundity compared to the wild progenitor Rr. However, due to its high overall number of fruits and seed production relative to both progenitors, CAwr was not negatively affected by granivory. Under conditions where granivory is excluded, CAwr has the highest relative fecundity compared to both progenitors , concurring with results from a prior study . The fecundity of protected Rr fruits was comparable to the high fecundity of protected CAwr fruits. A potential explanation for this result might be that Rr fruits detach easily from the mother plant. I found a large number of dropped Rr whole fruits relative to whole attached fruits . Finches perch when attempting to feed, with the result that many Rr fruits fall to the ground before they can be damaged on the branch.

Because the birds did not forage on the ground, the seeds in fallen fruits remained viable. Easily detachable fruits were also observed occasionally on CAwr plants but not as frequently as in the wild Rr progenitor . Fecundity values of the cultivated Rs lineage did not differ between unprotected and protected treatments. Rs exhibited low fecundity values overall, which is in agreement with other studies .However, the reproductive output of Rs may be even lower than reported because our measurement protocol likely led to an underestimate of the negative impact of the granivore for this lineage. Observations by us and other researchers indicated that birds open entire fruits of the cultivar Rs and consume all the seeds inside . Fully eaten fruits often appear similar to an empty pedicel and thus may not be counted as damaged at all. An additional caveat is that by planting the three lineages close together I may have influenced the granivore’s choice to feed more often on Rr and CAwr, which are bigger and more productive compared to Rs. Density-dependence has been observed in other studies of granivory and it is expected that birds make foraging choices to maximize their own fitness . Plant invasion often involves multiple introductions followed by a time lag during which invader populations overcome small population effects such as genetic drift and inbreeding causing Allee effect, Founder Effect, and bottlenecks . In addition, other mechanisms favor invasiveness like gene flow, potentially leading to interspecific hybridization, . Some invasive plants are a result of successful interspecific hybridizations, presenting defense traits that may be identical, different or additive relative to one or both progenitors, defining their susceptibility or resistance . Hybrids can be more susceptible: a comparison of the densities of herbivore and pathogen species on two progenitors species, Salix sericea and S. eriocephala and hybrid willow shrubs, revealed significantly higher herbivore densities on the hybrids . Althoughthe trend in the Fritz et al. study and others that he and collaborators reviewed do not support higher resistance in hybrids, other studies have found evidence for this. For example, the hybrid derived Helianthus annuus texanus acquired resistance to two types of herbivores from both ancestors H debilis and H annuus annuus increasing its relative fitness . In the case of invasive plants, hybridization can contribute to the evolution and success of invasive lineages . In addition, after establishment of long-standing populations in the community in a novel environment, invasive plants evolve and with them their potential enemies . And as it is the case for all ecological interactions, invasive species exist in a complex network, where new abiotic and biotic conditions imposing a new selective pressure on the plants that may cause an adaptive response from the plants . Considerable effort has been given to the idea that plant colonists have the advantage to be unknown by their new biotic enemies, i.e. herbivores, frugivores and pathogens. This idea, initially known as the “enemy release hypothesis”, was proposed by Elton in 1958 but can be traced back to Charles Darwin in 1859 and subsequently with an added evolutionary context became the Evolution of Increased Competitive Ability hypothesis by Blossey and Nötzold . The EICA predicts that invaders have the opportunity to evolve a competitive advantage compared to native taxa by re-allocating on plant growth instead of on plant defenses. Aspects of this hypothesis have been tested empirically multiple times at many levels, at both single sites and continent-wide scales,but the results have been equivocal .

With respect to pre-dispersal seed predation, procona valencia buckets previous studies have found that other factors are fundamental to maintain the demographics. For example, higher predation does not translate in lower fitness rates that will affect the population size or the following year’s adult density in invasive Centaurea solstitialis . Similarly, in spite of higher susceptibility to the effect of invertebrate pre-dispersal seed predation by the fly Hylemya sp. in natural and artificial hybrids crosses relative to progenitor species, Ipomopsis aggregata and I. tenuituba, flower traits and higher pollination rates compensate for seed losses . One of the fruit characteristics that has been found to reduce the negative effects of seed-eaters on fitness is multi-seeded fruits . In our study, estimated percentages of damage decreased when calculated based on estimated seeds removed compared to values based on fruit damage, diminishing the level of impact by the granivore. Thus, multi-seeded fruits reduce the effect of the granivore pressure on seed survival. Prior studies in non-invasive plants also support this idea; the impact of seed predation is reduced when plants produce multi-seeded fruits . To our knowledge, this study is the first to find evidence of the advantage of multi-seeded fruit in an invasive plant in the context of granivory. Aside from the density-dependent effect that may have distracted the birds from eating fruits of Rs, I propose that fruits of each lineage provide a different level of defense to the seeds inside. Currently it is known that fruit morphology does vary among the Raphanus lineages as does fruit mechanical defense . Invasive plants have been demonstrated to exhibit rapid evolutionary change suggesting that ~100 years of granivore interactions may have imposed substantial evolutionary pressure on the hybrid-derived fruits. This possibility coupled with the pre-existing hardened fruit wall in Raphanus raphanistrum leads to the hypothesis that fruit hardness is a genetically based trait that is favorably recombined as hybrids are formed and diversify in time . We never observed fully eaten fruits of California wild radish, but at most ~1/3 of the seeded portion. Previous work on CAwr mating system has found that the seeds inside an individual fruit are usually sired by various pollen sources and that the siring of the pollen occurs in a non-random manner , which has been related to a within-fruit seed size variation . The combination of these traits and the impact of the granivore provide a unique situation to study the combined effects of natural and sexual selection. To include the granivore’s perspective was beyond the scope of our study. Nevertheless, our observations are in agreement with other features of the physiological ecology of house finches. We only observed male house finches feeding on our experimental radish plants. According to Hill et al. house finches have a 6-month breeding season, which in their western distribution starts in January . The fruits in the experimental plants described here were fully formed in June, which is near the end of house finch breeding season and just prior to the onset of molting. Carotenoid based plumage pigmentation such as red, orange and yellow can only be acquired through diet and are known to have a significant function in sexual selection, immune system and senescence . To acquire the necessary pigments to color their plumage, male house finches need to include carotenoid rich food in their diet during the time the feathers are replaced and growing . Raphanus plants have carotenoid contents and are particularly rich in ?- carotenoids , which are the specific class of pigments that house finches most likely require to color their feathers . Female house finches prefer males with bright red plumage and the intensity of the red coloration in the males significantly correlations with: 1) an early beginning of the breeding season , 2) increase in number of brood for a given pair , as well as 3) the concentration and type of carotenoids in the food ingested . To our knowledge there is no study that has reported carotenoids extracted from radish seed endosperm or even fruit wall; most studies focus on cotyledons and leaves . However, carotenoid pigments in canola Brassica napus seeds, another Brassicaceae species have been reported . The pattern of fruit consumption described here represents a positive effect for house finches and,surprisingly, a positive effect for Raphanus. We propose a novel interpretation that granivore-fruit interactions in C. mexicanus- Raphanus are mutualistic. The results of our study provide information of an invasive hybrid derived lineage performance relative to both progenitor lineages in the context of a novel biotic interaction. The hybrid as well as the wild progenitor have a combination of traits that increases their ability to survive and persist in novel environments. High fecundity and multi-seeded fruits with mechanical defenses protect and compensate for the effect of the granivore. The wild progenitor has some advantages over the hybrid lineage at the predispersal seed predation phase. Although this result might be accurate I also suspect that our experimental design might have added some unwanted level of competition among the plants at the early growing stages affecting the hybrid performance .

The introgression region was split into 18 loci and a representative primer pair was made for each locus and these were sequenced using GT-Seq . Whether the region was from M82 or S. pennellii was determined by the presence of SNPs and can be found in Table S8. All F3 and F4 plants were grown in the greenhouse and compared to M82. The bHLH032 promoter region in S. pennellii is 200-bp larger than that of M82. This region was sequenced to determine if the lines contained the M82 or S. pennellii version of the gene and all data binned by genotypes determined by sequencing. Leaf shape for the select lines showed that those line containing the S. pennellii version of the bHLH032 had rounder leaves while those with the M82 version were closer to the M82 control although still significantly different . Likewise, the vascular density showed that those plants which had the S. pennellii bHLH032 had lower vascular density than those with the M82 version, which had a density the same as the M82 control plants . For both leaf shape and vascular density the heterozygous plants were more like the S. pennellii homozygous bHLH032 plants than the M82 containing plants . The BY for the S. pennellii and M82 bHLH032 containing plants was higher than the control M82 plants, suggesting that other parts of the introgression were still present and likely played some role in the increase in BY . The plants heterozygous for bHLH032 had a BY similar to the M82 control plants but did have a wider range of values. The trait measurements and the genotype of the bHLH032 gene present suggested that there was a likely role for bHLH032 in the regulation of BY through modulation of leaf shape and vasculature. To confirm this possible correlation, blueberry in pot we made bHLH032 CRISPR mutants in the M82 background.

Two RNA guides were made within the bHLH032 gene for CRISPR mutation, one in the first exon on the 5’ end of the bHLH domain and the second at the 5’ end of the second exon . We obtained ten CRISPR lines, however only the second guide in exon 2 was successful in creating mutations. Of these ten lines we chose lines 05, 06, and 10 to carry forward for further characterization. bHLH032-05 has a one base pair deletion near the guide site that creates a stop codon and truncates the gene early Similarly bHLH032-10 has a two base pair deletion which also creates an early stop codon, but it is located 20 amino acids further downstream than that found in bHLH032-05, and it alters the amino acid sequence between the guide site and the truncation stop codon . bHLH032– 06 was unique from the other mutations as it had a three base pair deletion which created a two amino acid deletion and one amino acid insertion . The T1 plants from these lines were grown up in the greenhouse and their leaf shape and vasculature characterized. All three lines had significantly rounder leaflets than that found in M82 and bHLH032-06 and bHLH032-10 had significantly reduced vasculature . bHLH032-05 did not have significantly lower vasculature but it was lower than M82 following the same trend as the other mutants . All three lines were carried forward after confirming a phenotype similar to BIL 260 , however bHLH032-05 also had fruiting/seed set deffect that caused it to have difficulty making fruit or seed. Because of this phenotype we decided not to continue with bHLH05 progeny and work only with bHLH032-06 and bHLH032-10, neither of which had seed set or fruiting issues. For bHLH032-06 and bHLH032-10 the T3 and T4 plants were grown in the greenhouse for characterization of BY along with the leaflet shape and vascular density. These plants are in the process of the being sequenced to determine if they have the same mutation as the T0 parents, however a few plants have been genotyped and they will be discussed here. bHLH032-10 lines that have been genotyped so far contain the two base pair deletion resulting in a stop codon /10.

Leaf shape analysis shows that none of these lines are significantly different from M82, however homozygous plants had significantly lower vascular density than that found in M82 or the heterozygous plants . Figure 5d shows example leaflets and vascular images all three genotypes, and while the homozyogous leaflet trends towards rounder than M82 it is still very similar. The vascular density of homozygous mutant plants was much lower, replicating the phenotype seen in BIL 260, despite not having round leaflets . The BY for the homozygous mutant plants was significantly higher than either M82 or heterozygous plants, indicating that the previously identified correlation between decreased vascular density and the total output of the plants holds in these lines . Thus, the pattern of inverse relationship between vascular density and BY seen in the original BIL 260 plants was replicated in the bHLH032-10 CRISPR plants .The primary focus of crop improvement, specifically yield, has been on improving photosynthesis as photosynthetic rates tend to correlate strongly with total biomass . However, more recent studies have shown that photo assimilates, when available in large quantities as would be found with high photosynthesis, are primarily converted to vegetative biomass in tomato as well as rice . Other characteristics such leaf shape were explored here as well as in previous studies, that revealed a potential correlation between BY and the shape of leaflets found in tomato . However, while BIL 260 and sub IL 4-3-4 showed a marked increase in BY and yield respectively, other lines such as BIL 267 and 139 also had rounder leaflets than that found in M82 but did not show an increase in BY . While models such as the PLS-Path Model seen in Rowland et al. indicate a direct relationship between leaf shape and BY, there are likely other genetic or physiological factors that confound this correlation. Photosynthetic rates in BIL 260 were slightly elevated over M82 and sub IL 4-3-4 which suggests that an increase in photosynthesis, as has been previously suggested, could result in increased BY as seen here . However, the large variability in photosynthesis rates creates some doubt as to the efficacy of this claim, as the BIL 260 photosynthetic rate is 27 µmols m-2 s -1 , compared to that for M82 at 25 µmols m-2 s -1 . The opposite, however, appears to be true with lower photosynthesis, and therefore lower photo assimilates, resulting in lower yields and BRIX values . All these data together suggests that while photosynthesis is necessary to increase BY there may well be a hard cap on how much it can improve crop output. If photosynthesis itself does not improve BY beyond a certain point, then research should investigate other areas such as sugar transport and storage. The sugar and starch concentrations in the leaves of BIL 260 and sub IL 4-3-4 show an increased mobilization of starch during night hours but no coinciding increase in sugar, unlike M82 where the same starch mobilization led to an increase in leaf sugar during the same time period . This points to mobilization and export of sugar as a likely reason for the increased BY seen in BIL 260 and sub IL 4-3-4. Leaf sugar in BIL 260 is lower than that for both other lines during the 1 to 5 am time period, plastic planters wholesale and correlates nicely to the fact that BIL 260 has a higher BY than either other line . How leaf shape relates to sugar export could be tied to the vascular density found in the leaves, as leaf shape and vasculature are closely linked during development .

The vascular density of BIL 260 is significantly lower than that of M82 or sub IL 4-3-4, indicating that a lower amount of vasculature per mm2 results in increased sugar export, a trend seen in previous studies . How decreased vascular density increases export and what is regulating these changes remain open questions. To address these questions, we performed WGS and identified three transcription factors introgressed from S. pennellii unique to BIL 260 . Of these three only bHLH032 showed up as DEG in RNA-seq data for SAM, mature leaves, and young leaves . Analysis of gene expression using dimensionality reduction methods, showed the Cluster 19 from young leaves was enriched in sugar related genes and contained the bHLH032 transcription factor. This suggests bHLH032 may be a regulator of sugar related metabolism, similar to the mobilization and export processes seen previously . bHLH032 is peripherally connected to the network in M82, which means it acts as an input or regulator to the network, working through a gene similar to the Arabidopsis genes BDX/DGR2 which regulate leaf and vascular development . bHLH032 is not present in the BIL 260 network, likely due to low expression levels precluding it from being an input into the network, and causing alterations to vascular development, leaf development through the tie to vascular development, and subsequent sugar export and processing .To identify if bHLH032 was an important part of this pathway we generated back crosses of BIL 260 with M82 to break up the introgression region, and indeed those lines in the F3 population that contained the S. pennellii version of bHLH032 had rounder leaves, lower vasculature density, and higher BY . These data strongly suggest that bHLH032 is a master regulator of these morphological characteristics which lead to increased BY. In fact, the CRISPR knockout mutants of bHLH032 in the M82 background show a similar trend, with lower vascular density and increased BY . Interestingly, the leaf shapes of these CRISPR lines were not different from M82, suggesting that the mutation in bHLH032 separated the normal link between leaf shape and vasculature, and pointing to vascular density as the driving factor behind increased BY . The decrease in bHLH032 causes changes both leaf shape or vasculature which correlate with BY directly . This also breaks the long known trend of BRIX and yield being inversely correlated to allow for increase in both. Thus bHLH032 could be an important regulator of BY, opening an avenue for tomato improvement. It and other transcription factors functioning in the regulation of leaf shape and vascular density provide a target for future research to continue tomato improvement.Gas exchange measurements were made in the field on attached leaves after the plants had recovered from transplanting. For the 2015 field season measurements were made weekly from week 10 through week 13 , week 14 through 17 , and 18 – 21 , on approximately 5 plants per week. For the 2014 and 2016 field seasons terminal harvest was performed on 60, and 20 plants, respectively. For all field seasons measurements were made on leaves from the upper and lower portion of the plants to eliminate positional bias within the plant and measured for three leaves per plant. The A , gst , transpiration, and ɸPS2 of a 6 cm2 area of the leaflet was measured using the LI-6400 XT infra-red gas exchange system , and a fluorescence head . The chamber was positioned on terminal leaflets such that the mid-vein was not within the measured area. Light within the chamber was provided by the fluorescence head at 1500 µmol m-2 s -1 PAR, and the chamber air flow volume was 400 µmols s-1 with the chamber atmosphere mixed by a fan. CO2 concentration within the chamber was set at 400 µmols mol-1 . Humidity, leaf, and chamber temperature were allowed to adjust to ambient conditions, however the chamber block temperature was not allowed to exceed 36°C. Measured leaflets were allowed to equilibrate for 2 to 3 minutes before measurements were taken, allowing sufficient time for photosynthetic rates to stabilize with only marginal variation.For the 2015 field season three plants per cultivar were destructively harvested each week. The final yield and fresh vegetative weight of each plant harvested, was measured using a hanging scale in the field. Five leaves were collected at random from the bottom and top of the plant to capture all canopy levels, and approximately nine fruit were collected for BRIX measurements.

In fleshy fruits, soluble sugars, including sucrose, fructose, and glucose, are not only essential for fruit growth and development but also central to fruit quality. Fruit taste and flavor is closely related to the composition and concentration of sugars and their balance with acids . As the composition and concentration of sugars at fruit maturity is determined by metabolic and transport processes during fruit development, understanding these processes and their regulation is important for fruit quality improvement. At the center of sugar metabolism in sink cells is the Sucrose cycle, previously named the Sucrose–Sucrose cycle or the futile Sucrose recycle, which consists of the breakdown of sucrose by invertase and sucrose synthase, the phosphorylation of the resulting hexoses and the interconversion between hexose phosphates and UDP-glucose, and the re-synthesis of sucrose via sucrose-6-phosphate synthase and sucrose-6- phosphate phosphatase. This metabolic system connects sugar metabolism with many other metabolic pathways such as glycolysis and tricarboxylic acid cycle, starch synthesis, and cellulose synthesis, and its coordination with the sugar transport system on the tonoplast is expected to determine the partitioning of sugars between metabolism in the cytosol and accumulation in the vacuole . In fleshy fruits, collection pot the concentration and distribution of sugars in parenchyma cells are affected via this cycle by developmental processes and environmental factors.

However, the biochemical regulation of the cycle and the associated transport system is not fully understood.Apple is one of the most economically important deciduous tree fruits worldwide. In apple and many other tree fruit species of the Rosaceae family, sorbitol is a primary end product of photosynthesis and a major phloem-translocated carbohydrate, accounting for 60–80% of the photosynthates produced in apple leaves and transported in the phloem. In source leaves, sorbitol is synthesized from glucose-6-phosphate in a two-step process: G6P is first converted to sorbitol-6-phosphate via aldose-6-phosphate reductase , then followed by dephosphorylation of S6P to sorbitol via S6P phosphatase. The loading of both sorbitol and sucrose into the companion cell-sieve element complex in the phloem is passive and symplastic in apple, but their phloem unloading in fruit involves an apoplastic step. Once released from the SE-CC complex of the phloem in apple fruit, sorbitol is taken up into the cytosol of parenchyma cells by plasma membrane-bound sorbitol transporters and then converted to fructose by sorbitol dehydrogenase ; sucrose is either directly taken up into parenchyma cells by sucrose transporters , or first converted to glucose and fructose by cell wall invertase and then transported into the parenchyma cells via hexose transporters. Compared with plants that transport and utilize only sucrose, such as Arabidopsis, tomato , and poplar , apple is unique in that both sorbitol and sucrose are transported in the phloem and are metabolized in sink organs. It is estimated that >80% of the total carbon flux goes through fructose in apple . Once taken up into parenchyma cells of fruit, both sorbitol and sucrose feed into the Sucrose cycle to meet the carbon requirement for fruit growth and development while excess carbon is converted to starch for storage in plastids or transported into vacuole by sugar transporters for accumulation.

Although we have characterized the genes and proteins involved in sugar metabolism and accumulation in apple, it remains unclear how apple trees adjust the Sucrose cycle and the transport system in response to altered supply of sorbitol and sucrose from source leaves. In transgenic apple trees with anti-sense suppression of A6PR, leaf sorbitol concentration is dramatically decreased, whereas sucrose concentration is significantly elevated in the source leaves, but neither leaf CO2 assimilation nor plant vegetative growth is altered. The decreased sorbitol synthesis leads to significant changes in the expression profile of key genes in leaf starch metabolism and many stress response genes. In addition to being a key metabolite in carbohydrate metabolism, sorbitol also acts as a signal regulating stamen development and pollen tube growth and resistance to Alternaria alternata in apple. In the shoot tips of the A6PR transgenic plants, both the activity and transcript level of SDH are down regulated, whereas those of sucrose synthase are upregulated in response to a lower sorbitol but higher sucrose supply. Teo et al. reported that fruit of the transgenic apple trees accumulated a higher level of glucose and lower levels of fructose and starch at maturity, but no significant difference was detected in the activity of key enzymes in sugar metabolism, CWINV, neutral invertase , fructokinase , hexokinase , or SPS between the transgenic lines and the untransformed control . Considering that anti-sense suppression of A6PR has drastically decreased leaf sorbitol level and increased sucrose level, leading to less sorbitol but more sucrose being transported in the phloem; and both transcript levels and activities of SDH and SUSY responded to the altered sorbitol and sucrose supply in the shoot tips of the transgenic plants, we predicted that the decreased supply of sorbitol and increased supply of sucrose would lead to down regulation of sorbitol metabolism and upregulation of sucrose metabolism in the transgenic fruit as well.

The discrepancy between the data obtained by Teo at al. and our predicted responses on the activities of sucrosemetabolizing enzymes in the transgenic fruit has prompted us to re-evaluate sugar metabolism and accumulation in the fruit of these transgenic plants to better understand how the Sucrose cycle and the sugar transport system respond to an altered supply of sorbitol and sucrose.Antisense suppression of A6PR significantly decreased sorbitol concentration but increased sucrose concentration while largely maintaining fructose and glucose concentrations in source leaves throughout fruit development in the two transgenic lines relative to the untransformed CK . Sorbitol concentration in the source leaves of anti-sense line A27 was decreased to ~70% initially and 13% at harvest of that detected in CK. For anti-sense line A04, sorbitol concentration was decreased to 32% initially and 10% at harvest of the CK level. By contrast, sucrose concentration in the source leaves of A27 and A04 was much higher than in CK throughout fruit development, 10 plastic plant pots with larger differences detected at later developmental stages . Concentrations of sorbitol and sucrose were also measured for source leaves, leaf petioles, and fruit pedicels at 75 days after bloom . Compared with CK, anti-sense lines A27 and A04 had lower concentration of sorbitol, higher concentration of sucrose, and lower molar ratio of sorbitol to sucrose in the source leaves, leaf petioles, and fruit pedicels. The abundance of sorbitol followed the order of source leaves > leaf petioles > fruit pedicels .Average fruit fresh weight did not differ significantly between the two anti-sense lines and CK during fruit development except for about a 10% lower value detected for A27 and A04 at 108 DAB and at harvest . Dark respiration was ~1.5–1.9-fold higher in A27 and A04 fruits than in CK fruits between 40 and 108 DAB during fruit development, but no significant difference was detected at harvest . Fruit yield per tree was significantly lower in the two anti-sense lines than in CK, largely due to lower average fruit weight at harvest as fruit number per tree was not significantly different between the two anti-sense lines and CK .Suppression of sorbitol synthesis in source leaves led to a significant decrease in sorbitol concentration in the fruit of two anti-sense lines A27 and A04 throughout fruit development, particularly in A04 . However, sucrose concentration was similar in the fruits of the two anti-sense lines and CK during fruit development with a higher level detected in the transgenic fruit only at 74 DAB. Fructose concentration showed no difference between the transgenic fruit and CK except being slightly lower at 108 DAB in the transgenic fruit. Compared with CK, concentrations of glucose and galactose were much higher throughout fruit development, with larger differences detected at later developmental stages. Concentrations of G6P and fructose-6- phosphate decreased during fruit development and were significantly lower in A27 and A04 than in CK from40 to 108 DAB . At fruit maturity , total soluble solids concentration was significantly higher in A27 and A04 than in CK . Fruit starch concentration did not show obvious difference between the transgenic lines and CK before 74 DAB but was slightly lower in A27 and A04 than in CK after 108 DAB .Our data clearly showed that sorbitol concentration was significantly lower, whereas sucrose concentration was significantly higher in the source leaves of 5-year-old transgenic “Greensleeves” apple trees with anti-sense suppression of A6PR compared with the untransformed CK throughout fruit development.

These results are consistent with those reported for the 1-year-old transgenic trees. The higher sucrose concentration in the source leaves is an indication that a larger proportion of the photo synthetically fixed carbon ends up in sucrose over a 24-h period because most of the starch accumulated during the day breaks down for sucrose synthesis at night in the transgenic plants although no difference in the carbon flux to sucrose during the day was detected. As both sorbitol and sucrose diffuse into SE-CC complex from mesophyll cells via plasmodesmata,transgenic plants. The lower concentration of sorbitol and higher concentration of sucrose in both leaf petiole and fruit pedicel and a smaller ratio of sorbitol to sucrose indicate that significantly less sorbitol but much more sucrose is translocated from leaves to fruit in the transgenic trees, which is consistent with a lower sorbitol but a higher sucrose concentration in the phloem exudates collected from fruit pedicels of these plants. The total amount of carbon translocated to fruit is expected to be very similar between the transgenic lines and the CK because all the trees had a very similar cropload and no significant difference was detected in average fruit dry weight between the transgenic lines and the CK at fruit maturity . These data clearly demonstrate that, when sorbitol synthesis is decreased in the source leaves, more sucrose is synthesized in the leaves and translocated to the fruit, thereby largely maintaining fruit growth and development. This is also consistent with the homeostasis of vegetative growth observed in the transgenic lines. The transgenic trees with decreased sorbitol synthesis grown under our experimental conditions were only slightly smaller after 5 years of growth than those of the untransformed CK . This is consistent with comparable photosynthetic rates measured in the transgenic lines and the untransformed CK throughout the growing season , with the lower rates detected only at fruit harvest being largely related to the leaf brown spots caused by Alternaria alternata in the transgenic lines. However, Teo et al. found that the transgenic trees were much smaller than the CK trees. This discrepancy is likely due to differences in growing conditions between the two locations. As sorbitol is implicated in drought-stress tolerance in apple, these trees might have experienced more drought stress under warm and dry conditions in California than under cool and humid conditions in upstate New York. In addition, as we strictly controlled cropload to a similar level each year by thinning flowers and young fruits, dry matter accumulation was not drastically different between the transgenic lines and the CK in our experiment.In response to a decreased sorbitol supply from source leaves, both the transcript level and the activity of SDH decreased in the transgenic fruit, which is consistent with previous findings in apple fruit cortex tissues fed with sorbitol and in shoot tips and fruit of the transgenic trees. As most of the fructose in apple fruit is converted from sorbitol by SDH, a significantly lower fructose level had been predicted in the transgenic fruit based on dramatically reduced import of sorbitol into the transgenic fruit and the associated lower SDH activity. However, the fructose level in the transgenic fruit was remarkably similar to that in the untransformed CK: no difference before 74 DAB and at harvest with only a slight difference detected at rapid fruit expansion between the transgenic fruit and the CK . This near homeostasis of fructose level in the transgenic fruit has clearly resulted from the response of the Sucrose cycle and the associated sugar transport system to increased availability of sucrose in the transgenic fruit, specifically, more sucrose is taken up into parenchyma cells in fruit after phloem unloading; more fructose is generated from sucrose breakdown by NINV and sucrose synthase and less fructose is phosphorylated by FK in the cytosol; and tonoplast sugar transporters are upregulated to take up more hexoses into the vacuole.

Significant IL’s were identified as those with a median transcript level .1 SD from the mean of all genes across all IL’s in the module.Differentially expressed genes with enriched transcript levels in laser-microdissected SAM/P0 versus P1 samples or hand-dissected samples of the SAM + P0-P4 or P5 sampled over developmental time were obtained from Chitwood et al. . Genes for which a differential expression call could be made were merged with detected eQTL using the merge function in R . For bootstrapping, cis- and transregulated transcripts were analyzed separately. Merged transcript abundance patterns were randomly permuted using the sample function against bin identity. For each test, 10,000 permutations were sampled to count the times that a particular transcript expression pattern was assigned to a bin more than the actual count. Resulting frequencies, representing a probability value, were multiple test adjusted using the BenjaminiHochberg method using p.adjust. Those bins with multiple test adjusted P values, 0.05 were analyzed further using visualizations created with ggplot2 .The rise of agriculture c. 7000 BC ensured a stable food supply, allowing human civilizations to develop and populations to grow . The challenge of feeding a growing population is exacerbated by climate unpredictability, with drought and temperature increases, leading to decreased crop yield . The narrow genetic base of most crops, combined with selection for performance under optimal conditions, has reduced the genetic variability in environmental stress responses, best indoor plant pots and the modern cultivars of tomato are no exception .

The wild relatives of tomato have the genetic ability to adapt to extreme habitats, and many heirloom cultivars also retain this ability as a result of directed breeding with wild species, and less selection for commercially valuable traits Heirloom tomatoes are defined as varieties, which have been passed down through multiple generations of a family . Improvement in tomato has focused on flowering, fruit traits, and disease resistance probably as a result of a perceived negative correlation between fruit size and sugar content . Thus, potential impacts of other factors on yield and fruit quality are relatively ignored . In a previous study by Chitwood et al. , a meta-analysis on a set of introgression lines linked leaf complexity and leaflet shape in tomato to fruit sugar content measured on the same lines by other researchers . This correlation showed that plants with complex and rounder leaflets also had increased fruit sugar content . Because leaves are the primary site of photosynthesis, it is possible that leaf shape changes may impact photosynthetic capacity and therefore result in different sugar content and yield in fruits. In addition to photosynthesis, sugar transport, and distribution to sinks are other potential sites of regulation in leaf function as source tissue. While sugar transport in plants is well described, distribution among different sink tissues is not fully understood . We analyzed tomato cultivars with varied yield and fruit quality, photosynthetic capacity, leaflet shape, and other vegetative traits and found that leaflet shape was strongly correlated to overall fruit quality assessed as a composite measure of BRIX and yield , with rounder leaflets positively correlated with higher BY values.

Photosynthesis, on the other hand, had a negative correlation with yield. Based on our analysis, leaf shape seems to play an important role in the distribution of photo assimilates. Additionally, we performed phylogenetic network analysis on 23 cultivars, including eight identified as having the rounder Potato Leaf Morph , known to be caused by a mutation in the C-locus , to determine their breeding histories and identify any potential selection for this trait.As photosynthesis is the primary source of sugar production in plants, a time course for photosynthesis, stomatal conductance , PARi and ɸPS2 was performed on all cultivars using a LI-6400XT . Additionally, we analyzed leaf sugar and vasculature for these lines in glasshouse conditions . Fig. 3 shows photosynthesis by gst and the trend is similar among all cultivars, with photosynthesis reaching a maximum rate after 0.6–0.8 gst, which is a standard response curve . Optimal photosynthetic performance regardless of light conditions has been observed in a forest tree species , and we saw the same at different levels of the canopy. Fig. 3 shows the PARi and ɸPS2. ɸPS2 had an overall downward trend across the season, as the amount of light used in photosystem II decreased with age. This corresponds well with the increase in vegetative biomass , and the increased PARi . Individual leaf contribution to overall photosynthesis, and therefore photons used in PSII, decreases as the leaf area of the plant increases. Because of this trend, we calculated the whole-plant photosynthesis, as photosynthesis/area, corrected for the green area visible in overhead images, and gst to capture the total rates and not just specifically measured leaves . The trend is linear for photosynthesis vs gst when the whole plant-exposed green area is incorporated, compared with Fig. 3 where the trend is more logarithmic. This corresponds to our previous observation that individual leaf contribution decreases as the total vegetative mass increases.

Leaf shape was shown to be strongly correlated with fruit BRIX and sugar accumulation in a metaanalysis of an introgression population . How leaf shape contributed to fruit BRIX was unclear, as shape and size of leaves do impact photosynthesis directly , but direct links between leaf shape and fruit quality appear lacking. Here, the heirloom population used displayed a wide array of leaf shapes, from very large and narrow to small and round. To understand if this range of leaf shapes had any impact on the overall fruit quality we measured leaflet shape and size for c. 3733 leaflets. Fig. 4 shows the resultant PCs of all primary leaflets measured and their relationship to traditional shape measures. PC1 contributes 78% of all variation found in the population and is tightly correlated with leaflet size , indicating that size was the largest source of variation among the heirloom leaflets . PC1 was also correlated with solidity , contributing to the slight shape changes seen in this PC . PC2 and PC4, while having no traditional shape measure correlation, indicate the left- and right handedness of the lateral primary leaflets, as these leaflets are mirror images of each other and therefore this measure describes the overall variation in leaf symmetry . PC3 accounts for 3.8% of all variation, but has a strong correlation with aspect ratio, or the width divided by the length of the leaflet, with an R 2 of 0.8 . PC3 therefore represents the roundness or narrowness of the leaflets, one aspect previously shown to be linked to fruit quality . The heirloom cultivars analyzed here were described as ‘potato leaf’, having broader, smoother leaves and typically lack the serration and lobes seen in other tomato varieties . However, blueberry container size despite this they had a wide range of leaf shape and size as illustrated in the leaf shape analysis . The classical potato leaf mutation is caused by a 5 kb transposable element inserted into the third exon of the C locus . To determine if this locus harbored mutations in the selected lines, a subset of the higher performing cultivars were selected for WGS analysis. Other mutations at the C locus have been described, and cause varied leaf shape . Fig. 5 shows the location of the mutations found in the C locus in these select lines. While the full Rider insertion could not be directly determined as the reference genome lacks this insertion, overhangs on reads in the third exon matched the Rider TE sequence . It is possible that different sizes and fragments of Rider are present in different cultivars, as the length and sequence of the overhangs varied . The identified Rider sequences were present in all but two of the sequenced lines, Prudens Purple and Glacier. No mutations were found in Glacier despite it having a rounder leaflets, although these were smaller in size with higher overall leaf complexity . Prudens Purple had a novel single base-pair substitution in the first exon outside the MYB/SANT conserved domain which results in the amino acid change P42R . We analyzed this mutation using the PROTEIN VARIANCE EFFECT ANALYZER , and found that it is predicted to be deleterious to the protein with a value of 8.454 , predicted to result in either a nonfunctional or partially functional protein . Based on leaf shape analysis, Prudens Purple shows a Potato Leaf like phenotype , although it differs slightly from the classical Potato Leaf shape seen in the reference allele and is reminiscent of the other mutations in C that have varying leaf shapes . These data demonstrate that different mutations in C, coupled with genetic background differences, may give rise to a range of leaf shapes seen among some of these cultivars.Pedigrees would probably inform the overall leaf shape in addition to the source of the C-locus mutations, but were not readily available.

To elucidate relationships among these cultivars we used the WGS data from the select cultivars as well as WGS data obtained from the 150 Genomes Project to assemble a phylogeny and perform phylogenetic network analysis . The phylogeny includes several commercial cultivars, commercial heirloom cultivars, Solanum pimpinellifolium, and Solanum lycopersicum var. cerasiforme. ABC Potato Leaf does not appear to cluster with other Potato Leaf heirloom cultivars analyzed here . Stupice, Glacier, and Bloody Butcher are closely related in this phylogeny, corresponding to their often being listed as closely related in popular literature , and congruent with phenotypic similarities they exhibit in fruit size and leaf shape. Bloody Butcher and Stupice both have the Rider insertion in the third exon at the C locus, while Glacier does not , suggesting the presence of other modifiers to leaf shape, which may have been selected for during the breedingof Glacier. A similar situation is seen in Prudens Purple , which is closely related to Jerry’s German Pink and Green Pearl. While Jerry’s German Pink and Green Pearl carry the Rider insertion at C, a novel single-base-pair substitution in the first exon leading to a deleterious effect on protein function is seen in Pruden’s Purple. Included in the clade is Silvery Fir Tree, a nonPotato Leaf heirloom with very distinct leaf morphology. These cultivars come from a similar region of eastern Europe , and our WGS phylogeny supports a regionspecific breeding history. The relationships between the Potato Leaf and nonPotato Leaf heirlooms are not well resolved in our WGS-based phylogeny, probably as a result of close relationships between the cultivars and interbreeding. To identify any breeding history specifically related to the Potato Leaf Morph, we performed PHYLONETWORKS analysis using the WGS SNPs . It is noteworthy that Prudens Purple with a unique mutation at the Clocus is not part of this series of hybridization events . These hybridization events suggest a breeding effort for desirable traits associated with this morphology. In addition we also analyzed chromosomes 1, 6 and 12 and found unique hybridizations for all of these chromosomes . These data suggest that analyzing a much larger group of tomato cultivars for hybridization history could be very fruitful.When doing large-scale field studies, it is difficult to understand how all the collected data points relate to each other, and what the causative relationships are . We performed several key correlation tests between measured traits , but to test all traits we would need to perform 91 independent correlation tests. As such, to decipher how all the physiological and morphological traits measured related to each other, we performed PLS-PM using SMARTPLS3.0 , which gives weighted causative paths with bootstrapping for confidence and significance values. In PLS-PM, each LV is a composite value of its associated MVs and forms an outer model . The inner model consists of the connections between LVs, with R 2 values indicating the degree to which each endogenous LV is described by the connections to it . Here, the only exogenous LV is leaf shape, as it has only its associated MVs and is descriptive of other LVs. Some LVs are described by other LVs within the model . When the value of a causative LV increases, the corresponding connected LVs change in accordance with their relationship with the causative variable.

Plant-associated Acinetobacter spp. have plant growth-promoting properties that include antagonism toward fungi and the ability to solubilize phosphate and produce the plant hormone gibberellic acid . Acinetobacter spp. are highly abundant in the floral nectar microbiome of Citrus paradisi and other plant species and were identified as a core member of the grapevine xylem sap microbiome . Their significant increase in relative abundance in the leaf microbiome at the time of flowering in citrus suggests a potential synergy between the foliar and floral microbiomes. Acinetobacter was also predicted to be a keystone taxon and was a major link between the flowering community and fruit set/development community clusters in our network. This enrichment in Acinetobacter may be simply due to selection imposed on the microbial community by the local plant environment, but it is tempting to speculate that Acinetobacter spp. provide an exogenous service to the plant by producing gibberellic acid and biologically available phosphorus to promote flowering that is in phase with its host’s phenological development. This hypothesis that the plant environment selects for taxa within its foliar microbiome that, in turn, promote its own reproductive growth warrants future inquiry. Specific bacterial enrichments also occurred at bloom time in grapevine, square plant pots further supporting the speculation of a microbial role in plant growth development that is in sync with plant developmental stage . We also observed signatures that indicate that specific taxa were depleted in relative abundance during flowering but enriched during fruit set.

Phylogenetic reconstruction of these taxa indicated that the majority of the taxa belonging to the Actinobacteria phylum were significantly depleted during flowering but subsequently enriched when trees begun to set fruit. This phylogenetic conservation of depletion/enrichment patterns within the Actinobacteria clade indicates that this is a nonrandom fluctuation within the microbiome structure associated with the transition from flowering to fruit production. As citrus trees set fruit, the fruits themselves begin exporting and importing hormones, such as indole acetic acid and cytokinins, respectively . This results in a change in hormone levels in leaves as well. Phytohormones can affect microbiome composition by being directly utilized as a carbon source or through other undefined mechanisms . We speculate that these hormonal shifts may place selective pressure on the foliar microbial community and lead to significant enrichments of Actinobacteria during fruit set and development. Specific differentially abundant taxa within the Actinobacteria clade that followed this pattern included Corynebacterium, Dietzia, Georgenia, and Ornithinimicrobium. Members of these genera can fix nitrogen and produce IAA, both of which are important supporters of fruit development . Thus, it is tempting to speculate that these taxa could play a role in coregulating fruit development in a manner that is synergistic with the host’s production of reproductive hormones. The biological role of Actinobacteria in the foliar microbiomes of plants is not well understood, but overall species richness was conserved across all phenological stages, except for fruit set, indicating that this phenological stage allows for microbial enrichments of specific taxa, particularly those belonging to the Actinobacteria phylum.

Genera outside the Actinobacteria clade were also depleted in leaves during floralbud development and full flowering, including Bacillus, Methylobacterium, Romboutsia, and Sphingomonas. Notably, Romboutsia and Sphingomonas were predicted to be keystone taxa in our microbe-microbe interaction network analysis, and all are in the top 20% highest betweenness centrality scores. Depletion of these taxa during flowering may have cascading effects that influence microbial species turnover by allowing other taxa, such as the Actinobacteria, to flourish during subsequent developmental stages, like fruit set. This suggests that microbial turnover in the foliar microbiome is mediated by selective pressures imposed by the plant developmental stage in conjunction with microbemicrobe interactions to modulate community diversity and composition. Further experimentation is needed to confirm these interactions. Microbial dispersal events can drive microbial turnover and influence the relative abundance of endogenous taxa in the community. Full flowering is a dynamic phenophase in plant development where there are frequent interactions between plants and pollinator species that rely on floral resources, like nectar and pollen. These macro-level interactions can also have effects at the microorganismal level. Pollinator visitation alters flower surface, nectar, and subsequent seed microbial community composition . During flowering, we observed striking microbial enrichments of bacteria taxa belonging to the Betaproteobacteria and Gammaproteobacteria clades that include Gilliamella, Snodgrassella, Bifidobacterium, and Frischella.

These enrichments in these anaerobic taxa were unique to the flowering phenophase and quickly declined following flowering, suggesting they are immigrants to the community and not endogenous members of the native microbiome. Moreover, these anaerobic taxa are prevalent in the bee gut microbiome . We hypothesize that these taxa immigrated into the citrus microbiome via a dispersal event during bee visitation. We consider this external influence host phenology-associated because phenophase-specific plant morphology regulate this diffuse interaction. Bacteria can be introduced to plants by bees and potentially migrate from the flower to the vascular bundles resulting in systemic movement within the plant . Leaf carbohydrate content is highest during flowering, which may promote the growth of these fermenting bacteria . Notably, Bifidobacterium was the only core leaf genus from the Actinobacteria phylum that was enriched during flowering, whereas the other 19 Actinobacteria taxa were depleted during flowering. This further supports the hypothesis that Bifidobacterium was introduced via a dispersal event and is not part of the endogenous microbiota like the other taxa in the plant-associated Actinobacteria clade. Nectar-inhabiting bacteria can influence nectar volatile profiles that, in turn, influence pollinator visitation preferences , and it would be interesting to determine if these putative immigrants contribute to shifts in nectar volatile profiles that affect bee feeding behaviors. The next frontier in microbiome research is to determine the functional roles that microbes play in microbe-microbe and host-microbiome interactions. Martiny et al. found that conservation of microbial traits was linked more strongly to vertical phylogenetic relatedness of the microorganisms within a microbiome than to traits that are shared among taxa by horizontal gene transfer . Similarly, we also observed phylogenetic conservation within microbial enrichments, suggesting that those groups of related organisms play similar functional roles during specific phenophases or across several phenophases. We speculate that taxa with high stability across phenophases may serve a community-stabilizing function, while low stability or phenophase-specific core microbes likely have more specialized, transient roles in the community. Because microbes can alter host phenology , which is a critical factor in plant health and productivity, incorporation of microbial presence/absence and patterns of enrichment into plant phenological models may improve phenophase timing predictions once the functional roles of these microbes are determined. This information could also lead to the commercialization of biofertilizers for horticultural purposes that could be applied at specific plant life stages to enhance crop productivity.Various genetically encodable reporters have been developed to monitor gene expression, protein subcellular localization, protein stability, hormonal signaling, and impacts of environmental signals. The green fluorescent protein and its derivatives such as RFP, mCherry, and YFP have many applications as reporters for gene expression or as fusion proteins. Although GFP is easy to use, plastic potting pots it needs light sources to visualize the fluorescence signals. The β-glucuronidase reporter has been widely used in plants for monitoring gene expression patterns and as a reporter for hormonal signaling. For example, DR5-GUS transgenic lines are commonly used to monitor auxin distribution and auxin signaling. Luciferase is another broadly used reporter in both animals and plants. Both GUS and luciferase require the addition of expensive substrates X-Gluc and luciferin, respectively. Whereas the traditional reporters have been very useful, they have limitations. Fluorescent proteins are often monitored under a microscope, rendering it less useful in analyzing plants in natural growing fields or analyzing large samples such as a tree. GUSstaining is invasive and often requires sacrifice of the plants. Luciferase can be used noninvasively, but it requires a special camera and spraying the expensive substrate. It is also not very practical to use them in fields.

GUS and luciferase may not be optimal for sterile conditions such as tissue culture because addition of substrates increases the chance for contamination of microbes. Therefore, there is a need to develop new reporter systems that can be widely used to monitor cellular activities noninvasively, continuously, and costeffectively. For the past few years, gene editing has been widely used in basic research and crop improvement. A visible marker for transgenes will greatly accelerate the isolation of edited plants that no longer harbor the gene editing machinery. Plants produce many colorful compounds that potentially can serve as reporters. For example, anthocyanins display bright red-blue colors and anthocyanin-producing rice plants have been used to generate interesting patternsin rice field. However, synthesis of anthocyanins requires multiple enzymes and varies greatly among different plants. It is difficult to use anthocyanin biosynthesis pathways as a universal visible reporter. Betalains are a class of plant natural products derived from the aminoacid tyrosine. The bright red color seen in beets, dragon fruit, Swiss chard, and other plants is resulted from accumulation of betalains. Biosynthesis of betalains has been well studied and only needs three enzymatic reactions to convert tyrosine into betalain. Tyrosine is first hydroxylated on the benzene ring, resulting in L-3,4-dihydroxyphenylalanine . The reaction is catalyzed by the P450 oxygenase CYP76AD1 . L-DOPA can be further oxidized into cyclo-DOPA by CYP76AD1 . Alternatively, LDOPA is catalyzed by L-DOPA 4,5-dioxygenase into betalamic acid, which is subsequently condensed with cyclo-DOPA into betanidin. The condensation reaction does not require an enzyme . Finally, a sugar moiety is added to betanidin by a glucosyltransferase to generate the colorful betalain . Betalain has a very bright red color, which potentially can serve as a reporter to track gene expression or to visualize transgenic events. Because every cell contains the amino-acid tyrosine, exogenous application of tyrosine to tissues may not be required. We hypothesized that betalain would be a more convenient reporter than the aforementioned reporters. It is visible to naked eyes without any needs for special equipment. It does not require processing samples and it allows continuously monitoring events throughout the life cycle of an organism. Moreover, it is applicable to large plants grown under normal field conditions. Herein, we synthesize an artificial open reading frame named RUBY that when expressed can produce all of the enzymes required for betalain biosynthesis. We show that RUBY is a very effective marker for noninvasively selecting transformation events in both rice and Arabidopsis. Moreover, we show that RUBY can be used to visualize gene expression without any chemical treatments or special equipment, providing useful tools for visualizing gene expression in large plants under natural field growth conditions.We transformed the 35S:RUBY construct into Arabidopsis using Agrobacterium-mediated floral dipping. Two days after floral dipping, we noticed that the transformed plants displayed patches of red color , indicating that the RUBY cassette was functionally expressed and that RUBY may be used to monitor transient Arabidopsis transformation. Once the seeds from the Agrobacterium-dipped plants were harvested, transgenic seeds could be easily differentiated from non-transgenic seeds . The transformed seeds had a dark red color , demonstrating that RUBY can be used as a visual selection marker for transgenic events in Arabidopsis. We previously used mCherry as a very effective marker to select transgenic events , which requires a dissecting microscope with fluoresence capability. RUBY is a better option because it does not require special equipment. The 35S:RUBY plants produced sufficient amount of betalain to become visually evident . Consistent with previous reports that CaMV 35S promoter is constitutively active, we observed red color in all tissues throughout the plant life cycle . We also expressed RUBY reporter under the control of the Maize UBIQUITIN promoter, which has been widely used to overexpress genes in monocots. Similar to 35S:RUBY plants, UBQ:RUBY plants were also visibly red in leaves, stem, and flowers . These results clearly demonstrated that RUBY could be expressed in Arabidopsis and that our RUBY reporter was able to functionally re-constitute the betalain biosynthetic pathway. We expressed RUBY using the seed specific At2S3 promoter, which we previously used to drive mCherry expression in Arabidopsis to facilitate the selection of transgenes. As shown in Fig. 2c, the transgenic plants were indistinguishable from wild type plants. When we checked the seeds in a silique from an At2S3:RUBY T1 plant, RUBY-expressing seeds displayed strong red color, whereas the non-transgenic seeds were green . RUBY can be conveniently used to select single T-DNA insertion events by analyzing the ratio of red seeds to green seeds, which should be ~3:1 for single insertions.

The molecular taste receptor, found in humans and rodents, responds to asparagine and aspartic acid . Asparagine is considered to serve as a nitrogen storage molecule and synthesized at night under low-carbon conditions . Asparagine and glutamate are synthesized from aspartate and glutamine through ASPARAGINE SYNTHETASE1 . Likewise, proline levels change in response to energy levels. PROLINE DEHYDROGENASE converts proline to glutamate . Recent studies demonstrate that S1-bZIPs directly regulate the expression of ProDH and ASN1 via binding to the C-boxes, ACT motifs , and G-boxes in their promoters, thereby influencing amino acid metabolism . Over expression of tbz17 mORF in tobacco significantly induces the expression of ASN, whereas silencing of tbz17 represses the expression of ProDH and ASN . One of the target genes of AtbZIP53 is ProDH2 . Over expression of SlbZIP1 and AtbZIP11 mORFs in the transgenic tomato and Arabidopsis significantly up-regulates the expression of ASN1 and ProDH2 and affects amino acid contents . For example, over expression of SlbZIP1 increases the content of alanine, aspartic acid, glutamate, serine, threonine, tyrosine, and total amino acid content. Energy deprivation induces the expression of ASN1 and ProDH, which contributes to the recycling of amino acids to mitigate deficits of carbon, nitrogen, and energy . Many amino acid catabolism related genes induced by AtbZIP11 are largely repressed by treatments with sugar . Moreover, under highsucrose conditions, the translation of AtbZIP11 is inhibited via a uORF . These findings indicate that ASN1 and ProDH are ultimately regulated in a sugar-dependent manner, blueberry pot with AtbZIP11 acting as the link between sugar signaling and amino acid/nitrogen metabolism .

Additionally, AtbZIP1 and AtbZIP53 are also involved in modulating amino acid metabolism during stress responses . In Arabidopsis, it has been demonstrated that AtbZIP53 preferentially forms heterodimers with group C-bZIP members like AtbZIP9, AtbZIP10, and AtbZIP25 for controlling the gene expression of ASN1 and ProDH . However, the interacting partners between the S1- and C-bZIPs are not identified in many other crops and need to be investigated in the future.Overexpression of S1-bZIP mORFs induces sugar-related gene expression and increases sugar content . Previous studies have shown that over expression of tbz17 and SlbZIP1 mORF up-regulates the expression level of genes encoding sucrose phosphate synthase and sucrose phosphate phosphatase , whereas silencing tbz17 down-regulates the expression of these genes . Furthermore, it has been demonstrated that constitutive expression of the S1-bZIP1s such as tbz17 and AtbZIP11 mORF significantly increases the sucrose concentration in transgenic lines . Interestingly, the contents of glucose and fructose were significantly increased and the citric acid content was significantly decreased in transgenic plants with over expression AtbZIP11 . The induction of the AtbZIP11 mORF also results in the up-regulation of genes associated with the metabolism of trehalose, myo-inositol and raffinose. Transgenic Arabidopsis lines over expressing AtbZIP11 showed decreased contents of the trehalose-6-phosphate , limiting the plant’s ability to use available sugars, thereby slowing plant growth. This growth inhibition in Arabidopsis cannot be reversed by the exogenous application of metabolizable sugars such as glucose and sucrose .

The use of the fruit-specific E8 promoter to drive over expression of SlbZIP1 increases the sugar contents in tomato while avoiding growth impairment . Remarkably, sucrose contents were approximately sixfold higher in transgenic lines with approximately 1.5-fold higher fructose, glucose, and total sugar contents than in wild type. Similar effects such as significantly increased glucose and fructose contents and significantly reduced citric acid content were observed in mutants with enhanced FvebZIP1.1 mORF protein expression due to the uORFs mutation . In a recent study, heterologous over expression of strawberry FvbZIP11 affects fruit quality and flavor in tomato . In comparison with wild type, the total soluble solid was significantly increased at the breaker, pink and red ripe stages in three transgenic tomato lines. The soluble sugar content was significantly accumulated at 30–50 days after anthesis in transgenic line 6. In addition, the titratable acid content was significantly reduced at 30 days after anthesis, while SS/TTA ratio was significantly increased from 20 to 50 days after anthesis in the transgenic tomato line . Taken together, these studies demonstrate that the S1-bZIPs play important roles in the regulation of sugar metabolism for quality improvement in plants.S1-bZIPs play an essential role in plant adaptation to unfavorable conditions . It has been documented that S1-bZIPs play important roles in plant innate immunity, especially against attack by various pathogens , and in response to abiotic stresses, such as cold , drought , and salinity . It has been demonstrated that the C-/S1-bZIP-SnRK1 complex participates in the reprogramming of primary metabolism related to carbohydrate and amino acid and induces salt stress tolerance through ABA-independent signaling in Arabidopsis roots .

Similarly, C-/S1-bZIP-SnRK1 signaling is involved in defenses against biotic stresses, which are also energy-consuming processes that require metabolic readjustment in plants . Research in our laboratory has suggested that petunia PhOBF1, a homolog of AtbZIP11, is involved in plant defenses against a wide range of viral pathogens . In the study, silencing PhOBF1 resulted in the reduction of RNA silencing-related gene expression, including RNA-dependent RNA polymerases, Dicer-like RNase III enzymes, and Argonaut. PhOBF1-RNAi plants displayed a compromised resistance to tobacco rattle virus and tobacco mosaic virus . On the other hand, over expression of PhOBF1 in petunia enhances resistance to these virus infections. Interestingly, PhOBF1-silenced petunia lines produced much lower levels of the compounds associated with the shikimate and phenylpropanoid pathways such as free salicylic acid , salicylic acid glucoside, and phenylalanine, but much higher levels of those were detected in PhOBF1 over expressing plants . Intriguingly, PhbZIP44, a paralog of PhOBF1 appears to be unable to participate in this antiviral process, suggesting functional diversity and specificity among the S1-bZIPs . In Arabidopsis, S1-bZIPs AtbZIP11/ATB2, AtbZIP44, AtbZIP2/GBF5, and AtbZIP53 can bind to a 6-bp cis-acting element located in the promoter of ProDH , which is responsive to hypoosmolarity and proline. AtbZIP53 directly and strongly promotes hypoosmolarity induced transcription of ProDH, which is enhanced by the synergistic interplay between AtbZIP53 and the group C member AtbZIP10 . Analysis of transcriptome data has revealed the complexity of the response to abiotic stresses by S1-AtbZIPs. For instance, the transcript level of AtbZIP53 was found to be strongly up-regulated by salt stress in roots and by osmotic stress in green tissues. Cold, osmotic, and salt elicitors were found to remarkably increase the expression of AtbZIP1 in roots and AtbZIP11 in green tissues but inhibit the expression of AtbZIP2 in green tissues. AtbZIP44 shows a solid and specific response to cold stress in the root and to salinity in green tissues . The expression of AtbZIP1 in roots was significantly induced by salt treatment. Arabidopsis bzip1 bzip53 double mutant reprograms carbohydrate and amino acid metabolism to help roots adapt to salt stress. Furthermore, AtbZIP1 binds the promoter of BCAT2 and TAT7 and plays a role as a signalling module of SnRK1-bZIP1 under salt stress. This pathway is independent of ABA-SnRK2-AREB signaling pathways, whereas bZIP53 transcription partially depends on the SnRK2/AREB pathway . In tomato, SlbZIP1 increases salt tolerance by increasing the gene expression related to ABA biosynthesis and signal transduction . In response to water deficiency, two cucumber S1-bZIP member transcripts accumulated in the root but decreased in leaves . Likewise, in sweet potato, the expression of IbbZIP1 is highly induced by treatments with NaCl and ABA. Abiotic stress-related genes are significantly up-regulated in the transgenic Arabidopsis over expressing IbbZIP1, suggesting the role of IbbZIP1 in salt and drought tolerance . In apple, an S1-bZIP, MdbZIP80, has been shown to negatively regulate cytokinin-mediated drought and salt tolerance . This study shows that MdbZIP80 specifically heterodimerizes with C-bZIPs MdbZIP2 and MdbZIP39. The formed C-/S1-bZIP complex then binds to the ACTCAT motif in the promoter of MdIPT5b, a gene encoding the rate-limiting enzyme isopentenyltransferase in the cytokinin biosynthesis pathway, thereby suppressing its expression. This leads to drought and salt stress response through the cytokinin pathway by delaying drought-induced premature leaf senescence by reducing oxidative damage and maintaining plant growth . Another study demonstrates that low temperature stress induces mlipl5 expression, nursery pots and the protein subsequently binds to the promoter region of Adh1 . Interestingly, mechanical damage in tea leaves leads to the activation of S1-bZIPs such as CsbZIP2, −11, −14, −16, −20, −21, −28 and −30 . Overall, it appears that the expression levels of these S1-bZIPs respond to stress signals in a tissue-specific manner. The members of S1-bZIP share partially redundant functions but play a role in unique regulatory mechanisms. Generally, the S1- and C-AtbZIPs heterodimerize to mediate stress signal transduction cascades. For example, S1-bZIP AtbZIP53 forms heterodimers with group C-bZIP members such as AtbZIP10 or AtbZIP25 and increases DNA binding activity, resulting in strong activation of the target genes. These heterodimers can also form tertiary complexes with the non-bZIP protein ABI3 to play a synergistic role in target gene expression ; however, it needs to be demonstrated whether other members of S1-bZIP such as AtbZIP1 heterodimers are formed under stress conditions .

The S1-bZIP gene low-temperature-induced protein 19 is significantly induced by low temperature in monocots . The LIP19 protein appears to be unable to form homodimers and bind to DNA in rice . However, the counterpart of LIP19 proteins in maize and wheat can form homodimers and bind to cis-elements in DNA sequences . The WLIP19 can heterodimerize with wheat TaOBF1, another low temperature-responsive S1-bZIP member. The stable heterodimerization between LIP19-type and OBF1-type proteins seems to induce the expression of target genes in response to different abiotic stresses, especially cold stress . However, there is no definitive evidence showing that the formation of heterodimers or homodimers between WLIP19 and TaOBF1 directly affects the expression of the downstream stress-responsive genes including COR and LEA genes . Recent research indicates that a group C-bZIP TabZIP6 dimerizes with WLIP19, TaOBF1, or itself and then binds to the promoters of genes encoding CBFs , resulting in inhibition of their expression. These dimers can also inhibit the expression of some COR genes . Rice S1-bZIP plays a vital role in ABA-mediated drought and salt stress response. One of the S1-bZIPs, OsbZIP71, appears to be able to form homodimers and heterodimers with group C-bZIP members OsbZIP15, OsbZIP20, OsbZIP33, and OsbZIP88. It has been speculated that these heterodimers help OsbZIP71 bind to the promoters of its target genes, OsNHX1, and COR413-TM1 because OsbZIP71 on its own has weak DNA-binding activity to the G-box element and no transcriptional activation activity . Thus, the interplay between C-group and S1-subgroup is proposed to affect plant response to stress.Plant growth and development are tightly interlinked with the control of metabolism, especially energy homeostasis. Transient energy deprivation causes plants to adjust their metabolism to adapt to daily light/dark cycles and unpredictable environmental changes. It has been proposed that the Snf1- related kinase 1 and Target of Rapamycin kinase function to reprogram plant metabolism in response to the energy status . Evidence suggests that SnRK1 mediates the phosphorylation of S1-bZIPs to control plant growth and development under starvation and nutrient-replete conditions . As the transcriptional regulators downstream of SnRK1, AtbZIP11 can directly control a subset of SnRK1-dependent genes via binding to G-box elements in their promoter regions . Furthermore, heterodimerization between group C- and S1- bZIPs is enhanced by the phosphorylation of group C-bZIPs by SnRK1. Phosphorylation of AtbZIP63 provides the structural basis for forming the AtbZIP63-AtbZIP1-SnRK1/AtbZIP63- AtbZIP11-SnRK1 complex and ultimately leads to the adjustment of metabolism to ensure plant survival under low energy conditions . Notably, the formation of the complex is dependent on the SnRK1-specific phosphorylation sites, which are pivotal for the function of AtbZIP1 and AtbZIP53 . Additionally, the identification of many SnRK1-independent genes regulated by AtbZIP11 indicates a function of AtbZIP11 beyond SnRK1 signaling . It seems that heterodimers within the C-/S1- bZIP network function as a hub to integrate SnRK1-dependent and -independent signals to adjust growth/development and stress responses . Recent studies showed that S1-bZIPs regulate the root apical meristem size through controlling polar auxin flux . Under low energy conditions, AtbZIP2, AtbZIP11, and AtbZIP44 directlyactivate the transcription of INDOLE-3-ACETIC ACID PROTEIN 3/SHORT HYPOCOTYL 2 , a negative regulator of auxin signaling, which leads to the down-regulation of PIN-FORMED genes, limiting polar auxin transport to the root tip and blocking auxin-driven primary root growth .

Plant microbiomes are dynamic and undergo successional changes with plant development, possibly with new introductions occurring throughout the plant life cycle. Several bacterial reservoirs for the phyllosphere microbiome have been reported, including the air, insect pollinators, seed, other nearby plants, and meteorological conditions. Te impact of the latter on fresh produce crop microbiomes is of particular interest due to the highly variable nature of weather-related events, variation due to geography, and anticipated changes in precipitation patterns in the coming years due to climate change. Increased precipitation and humidity often favor the development of plant disease. Similarly, the prevalence of several foodborne pathogens including pathogenic Escherichia coli, Campylobacter jejuni, Salmonella enterica and Bacillus cereus has been correlated with elevated environmental temperature and humidity. In trials assessing the fate of E coli, fecal coliforms and enterococci applied to the lettuce phyllosphere, bacterial decline rates were slower under moderate and regular rain patterns. At the community level, rainfall events may coincide with drastic changes in the leaf surface microbiomes of canola plants, although changes due to plant development could be difficult to detangle. Below ground, some soil microbial communities are influenced by drying and wetting frequencies, especially those not normally exposed to large fuctuations in soil moisture. Rain may shift the microbial profile of phyllosphere communities through direct seeding of microbes present in rainwater, growing blueberries in pots splash from surrounding soil, increasing water availability for existing microbes, or by washing of loosely adhered epiphytes, creating opportunities for others to fill their former niche.

Airborne biological particles, including bacteria and fungi, may act as ice or cloud nuclei, particles around which rain droplets form. Levels of bioaerosols are elevated during rain events, and in fact, plants have been suggested as “cloud seeders”. Airborne microbes, classified as bioaerosols, may be transferred to plant surfaces directly via rainfall or indirectlyfrom standing water after rainfall. In fact, Salmonella enterica serovar Typhimurium is capable of aerosolizing from puddles and colonizing tomato plants following simulated rain events. Rain splash dispersal can facilitate the transfer of human enteric bacteria from bulk soil to leaf and fruit surfaces even with the use of plastic mulch as a barrier. Other microbes including plant pathogens are similarly capable of aerosolizing and retaining viability, sometimes incorporating aerosolization as part of their lifecycle. In the face of a changing and more variable climate, managing crop protection to ensure crop diversity, food security and food safety will necessitate a deep understanding of crop systems including their association with microorganisms and how they respond to external conditions and stresses. To garner a more comprehensive understanding of the impact of rain on the phytobiome of fresh produce crops that are vulnerable not only to plant disease but also colonization by human pathogens, we characterized the epiphytic bacterial communities dwelling on two commercially important fresh produce crops. A temporal assessment of the epiphytic bacterial communities of commercially cultivated cucumber fruit following a rain event, and tomato carpoplane and leaves surrounding two rain events was conducted.Sterile deionized water was added to sample bags .

Submerged samples were hand massaged through the bag for 30 s then sonicated in a Branson Ultrasonic Bath 8510 for 3 min at a frequency of 40 kHz to dislodge bacterial cells from the carpoplane and phylloplane. Samples were hand massaged again and sonicated for an additional 3 min before filtration. Carpoplane and phylloplane washes were filtered through sterile 0.22 µm nitro-cellulose filters , and filters were frozen at −80 °C until further processing. Total community DNA was extracted from filters using the MoBio PowerWater kit . Te V1-V3 region of the 16 S rRNA gene was chosen for use in bacterial community profiling using 8F-533R primers. Sequencing was carried out using 300-bp paired-end sequencing on the Illumina MiSeq . Illumina’s protocol for 16 S Metagenomic Sequencing Library Preparation was followed for all samples as previously described.Quality filtering and sequence analysis were carried out using QIIME v. 1.8, Mothur v. 1.34, and Phyloseq v. 1.24.0 in R v. 3.5.0. Prior to alignment, sequences went through several quality filtering steps to remove chimeras, non-target sequences , and sequences less than 100 bp in length. Sequences were aligned to the Greengenes Core Set using PyNAST, and taxonomy assignment utilized the RDP Classifier 2.2. Reads that failed to match the reference database were clustered de novo using UCLUST v. 1.2.22. To ensure comparability between samples, the dataset was subsampled to the lowest common sequencing depth, 8,200 sequences per sample. Sample types were analyzed separately to assess the influence of rainfall events on bacterial diversity for each of these niches. Beta diversity was assessed using both unweighted distance matrices and matrices weighted by relative taxon abundance. Phylogenetic distance was incorporated into both distance matrices using UniFrac.

Bray-Curtis dissimilarity, which includes abundance but not relatedness in calculation of dissimilarity, was also assessed. Adonis , a non-parametric MANOVA from R’s Vegan package, was implemented to assess significance of treatment influence on bacterial community structure. Using Principal Coordinates Analysis generated through R’s Vegan and Phyloseq packages, plots were created to visualize β-diversity. Alpha diversity was assessed on the rarefed dataset in Phyloseq using both Observed OTUs and Shannon Index metrics. Observed OTUs represent the number of unique Operational Taxonomic Units at 97% sequence similarity, while the Shannon Index takes into account the proportional abundances of the observed OTUs. To compare α-diversity between groups of samples, ANOVA was employed followed by Tukey’s HSD tests for pairwise comparisons, in R’s stats package v. 3.5.0. More than 95% of sequences were identified to the family-level and differential abundance analysis was continued at this taxonomic rank for the unrarefed dataset using DESEQ. 2. Sample information, sequence data and contingency matrices are available with Qiita study id 12262 . Sequence data have been deposited in the European Nucleotide Archive at the European Bio-informatic Institute under accession number ERP 118277.Daily precipitation measurements were obtained from the National Oceanic and Atmospheric Administration website , using climatological data collected from a weather station located 9km from the sample site. Only limited weather data was available from the local station, so additional weather measurements, including temperature, barometric pressure, and wind speed, were acquired an Automated Weather Observing Station located 18 km away. Te weather station is operated by the Federal Aviation Administration and administered by NOAA , and data was accessed through Weather Underground .Sequencing metrics. Approximately 4 million raw reads from 94 multiplexed samples with an average length of 463 bases, and an average Q score of 35 were further filtered for quality. Reads 1 and 2 were merged at an average efficiency of 83%. High quality unmerged read 1 was also included in downstream analysis. All samples had a Good’s Coverage value exceeding 0.95, indicating that samples were sequenced to a level nearing saturation. After rarefaction, 770,810 sequences were retained for the final analysis. Weather. A dry period had been recorded prior to the commencement of sampling, with the most recent precipitation dating back to a 21mm rain event on 8/20, 3 weeks prior to the first pre-rain sampling. The first rain event on 9/12 recorded 9.14mm of precipitation and the second event on 9/21 reached 9.65mm . The highest daily temperature was recorded on 9/9 and a wind gust occurred on 9/13 around sampling time. Barometric pressure was low on 9/9 and 9/13 compared to the other sampling dates. Rain 1 was accompanied by thunder and lightning. Cucumber carpoplane. Following the 9/12 rain event , a significant change in cucumber fruit surface bacterial community structure was observed for both unweighted UniFrac and weighted UniFrac analyses . Cucumber fruit samples collected 3 days prior to Rain 1 supported bacterial communities that clustered separately from those collected 1 day after Rain 1, drainage gutter and the largest average unweighted UniFrac distances were obtained between samples collected on 9/9–9/13 and 9/9–9/17 . Although some samples collected on 9/17 generated bacterial community profiles resembling the pre-rain profile, some resembled the immediate post-rain profile, a trend evident across 3 different distance metrics . Therefore, within 4 days a partial return to the pre-rain profile was detected. On the cucumber carpoplane, α-diversity increased significantly following Rain 1, as measured by both Observed OTUs and Shannon Index , escalating from an average of 232 to 310 OTUs per sample, a 33.6% increase . Five days after Rain 1, α-diversity remained elevated compared to pre-rain levels , with an average of 300 OTUs per cucumber.

Many of these OTUs were introduced across all replicates, indicating a common source. A core microbiome analysis was conducted to identify taxa shared by 100% of samples collected on each date and across multiple dates. Thirty-eight OTUs were ubiquitous among all dates. Seventy-four OTUs not observed in pre-rain cucumber carpoplane samples were identified in samples collected 1 day post-rain . Of these, 35 OTUs were retained across all replicates 4 days later. By contrast, only 7 OTUs observed in pre-rain samples were not detected 1 day post-rain, with 3 of these being observed again 5 days post-rain. In addition to the introduction of new taxa, changes in the relative abundance of established taxa on the cucumber surface were observed following Rain 1 . Of 809 total OTUs in the cucumber dataset, 112 were differentially abundant between samples collected on Sept 9 and 13 . At the family level, 16 bacterial families differed between the pre- and post-rain time points. Notably, the family Xanthomonadaceae increased from an average of 1.2% to 9.6% relative abundance following rain , dropping to 2.4% 5 days after rain. The Oxalobacteriaceae exhibited a similar increase, from 0.6% to 7.0% , but in this case average relative abundance remained high after 4 days, at 7.1%. Similarly, the Sphingobacteriaceae and Comamonadaceae, initially detected at less than 0.2% average relative abundance, increased at least an order of magnitude in relative abundance following rainfall, remaining elevated 4 days later . Relative abundance for several of the most dominant bacterial families on the cucumber surface declined or increased following rainfall. Te Sphingomonadaceae decreased from an average of 9.6% to 5.1% relative abundance following rainfall but increased 4 days later to 7.6%. Similarly, the average relative abundance of the family Microbacteriaceae diminished following rainfall , increasing to an even higher average relative abundance later . The Enterobacteriaceae demonstrated an opposite shift that was not significant , increasing 1 day after rain from 18.4% to 21.9%, later returning to 17.2% average relative abundance . Although these changes in relative abundance are indicative of community shifts, they do not necessarily translate to increases or decreases in the absolute abundance of certain taxa. This may be the reason why β-diversity of the tomato carpoplane did not strictly parallel the same trend observed on cucumber fruit. There was an overall effect of sampling date on bacterial communities when analyzed using unweighted UniFrac distance and Bray-Curtis dissimilarity but not weighted UniFrac distance . Throughout the sampling period, unweighted UniFrac distance increased steadily in comparison to the first pre-rain sampling date, with the greatest distance measured between samples collected on 9/9 and 9/25 . On the other hand, following Rain 1, α-diversity on the tomato carpoplane resembled the dynamics seen on cucumbers. Observed OTU count increased from 185 to 251 OTUs per sample from 9/9 to 9/13 . Five days after Rain 1, α-diversity by both measures was indistinguishable from pre-rain and 1 day post-rain levels , at 231 OTUs per sample. Following Rain 2, observed OTU count remained elevated compared to pre-Rain 1 but did not significantly increase beyond 9/13 levels. Analysis of the same dates by Shannon Index suggests that across the sampling period, α-diversity was different only between 9/9 and 9/13 . Following Rain 1, similar trends in the core microbiome as those on the cucumber carpoplane were observed on tomato fruit. For the 3 dates surrounding Rain 1, tomato fruit collected on each day supported OTUs common to all samples collected on that date but not observed on any other date, with 9/17 hosting the most unique core OTUs . Moreover, 42 additional OTUs were present across all post-Rain 1 samples, of which 18 persisted in all samples 4 days later. Following Rain 2, only 17 new OTUs were detected on all tomato fruit samples, 7 of which remained present on all samples on the final sampling day.

Deer mice typically have a small home range , so if the target species for your control program is the deer mouse only, you should use a 98-foot spacing. This ensures that any deer mouse will have access to at least one bait station within its home range. Roof rats have a larger home range , so the 164-foot spacing will put at least one bait station in each rat’s home range while reducing the total number of bait stations required to effectively treat the orchard. If both roof rats and deer mice are present, use the 98-foot spacing.These formulas will calculate the number of bait stations required for the orchard and also provide an approximate location for each bait station. Note, however, that the actual spacing between individual trees and rows of trees will dictate the ultimate placement of each bait station, which will in some cases be in the tree that is closest to the calculated location. Use bungee cords or wire to attach to bait stations to tree branches. Bungee cord takes less time than wire; wire is cheaper, but it is also harder to use and must be frequently replaced. Nylon cable ties were not effective for keeping bait stations securely attached to branches. To prevent spillage, bait stations should only be attached to branches that are at an angle of 45° or less from the main trunk. Bait stations can be attached to the top or the underside of the branch, square pot but must be rotated so the hole in each end cap is at the top . An initial amount of one cup of bait should be added to each bait station. Check the bait stations on a regular basis to ensure a constant supply of bait; you can adjust the amount of bait you put in each station according to uptake. If necessary, you can put as much as 1 pound of bait per station at one time.

It is important to note that diphacinone is a first-generation anticoagulant that requires multiple feedings to give the target species a toxic dose. As such, it is essential that you maintain a constant supply of bait in the stations throughout the duration of the baiting process. You can deploy bait stations without bait for a few days at first to allow rodents to become acclimatized. After this initial period, keep replenishing the bait for around 4 weeks or until consumption stops. After cessation of a baiting program, you can keep the bait stations in place, but filled with non-toxic oats, in order to assess re-invasion by nearby roof rats or deer mice. If you do see evidence of re-invasion, you can continue baiting until the start of the growing season.Bait stations may also be deployed at ground level, but previous studies have found that elevated baits are more effective against roof rats than bait placed at ground level . Additionally, bait stations placed at ground level increase the likelihood that non-target animals will fall prey to the bait. Re-invasion rates can be high for many rodent species, with immigration occurring as soon as two months after control if adjacent areas are home to large populations . As such, it is important to consider rat and mouse outbreaks and invasions at a broader, landscape level. Coordinated control programs implemented by neighboring landowners should provide the best results against damaging rat and mouse populations.In wet and humid conditions, the bait can absorb moisture. If this occurs, replace it with fresh, dry bait to maintain efficacy. Bait stations were designed to minimize spillage of bait during rodent feeding, but you still need to monitor for bait spillage and immediately clean up any spilled bait.

Deer mice have been known to nest in bait stations. Deer mice are known reservoirs of the Sin Nombre virus, which causes the potentially fatal Hantavirus Pulmonary Syndrome . This virus is transmitted through the inhalation or ingestion of aerosolized saliva, feces, or urine of deer mice. Because of this, it is important to make sure that any wind is behind the bait application technician when he or she opens the bait stations, so that any aerosolized particles will be carried away, downwind. Although concerns of secondary toxicity are typically quite low with first-generation anticoagulants , they still hold some inherent risk. As such, the survey of pest carcasses is always an important part of implementing a baiting program using anticoagulants. During a baiting program, most rodents die below ground but a few will be found above ground. Check for above ground pest carcasses once or twice a day. Remove the dead rodents and dispose of them by burying or burning . Alternatively, you can wrap carcasses in newspaper or double-bag them and dispose of them in the trash. Latex or nitrile gloves should be worn when handling dead rodents to prevent potential infection from the various diseases or parasites they may carry. Initial start-up costs for an effective baiting program will vary depending on which pest species are present, since deer mice require more bait stations per acre than roof rats alone. At the time of this writing, the individual bait stations cost $4.72 each, and 1 pound of the 0.005% diphacinone oat bait cost $1.75. Estimated start-up costs, then, using the 164-foot spacing, would be approximately $624.07 per 80-acre orchard . Initial costs for deer mouse management would cost considerably more: $1,794.17 per 80-acre orchard . These estimates are based on initial placement of one cup of bait per bait station. If rates of infestation are high, you may need to place additional bait. Also, these estimates do not include tie-up or labor costs, which will vary considerably depending on the tying method you use to fasten bait stations to tree branches.

It is important to note that subsequent costs for bait programs go down dramatically, since the initial cost of bait stations accounts for the bulk of the expense of a start-up baiting program. The bait stations described are very durable and can be used across multiple fields and multiple years, further reducing the long-term cost of baiting programs. Ultimately, we believe this baiting strategy will allow for effective, economical management of roof rats and deer mice in nut and tree fruit orchards in most areas where these species are found. This approach also substantially reduces the potential for negative impacts from rodenticide bait on non-target species, thereby minimizing the hazard to the natural environment.There is strong evidence that the majority of wild and cultivated plant species benefit from or rely entirely on the transportation of pollen grains by bees, other insects, birds and mammals . Furthermore, at the within-plant or whole plant scales, many crops exhibit decreased crop production in response to decreases in the number and types of pollinator , and these results have been extrapolated to field and landscape scales , as well as regional and global scales . In natural populations, pollination may also strongly limit reproduction, particularly in fragmented landscapes . While pollination clearly affects reproductive yields of many wild and crop plants , the degree to which it regulates yield in real-world cropping systems is debated . The emphasis on the role of pollination in determining yield has been criticised on the basis that it does not account for possible post-pollination processes affecting the amount and quality of fruit maturation . For example, water and nutrient limitation can strongly affect early fruit abortion and variation in losses to pests and diseases may ultimately be more important than pollination in determining realized yields . Furthermore, the interactions between pollination and post-pollination processes are rarely considered for wild plants and are almost unstudied in crop plants . Given widespread concerns about colony losses of Apis mellifera L., a major crop pollinator worldwide , as well as evidence for declines of other pollinators at landscape and regional scales , a greater understanding of the importance of pollination processes for reproduction and yield is critically needed. To date, studies of pollination limitation in tree crops have examined only a subset of flowers per plant or invokepollinator limitation without supplementary hand-cross pollination of the flowers . Instead, square plastic planter yields should be analysed at the whole plant scale to avoid the confounding effects of resource allocation among individual flowers or branches . Thus, experiments are needed that compare hand-pollinated yields to those in which pollinators are totally excluded , with and without resource applications. Furthermore, high fruit yield may come at the expense of the trees’ vegetative performance, such as quantity of foliage. The indirect influences of pollination in combination with plant resource variation on vegetative features of crops such as leaf quality and quantity is not yet investigated. Commercial almond production is an excellent model system for testing effects of pollination on crop production and foliage variables in different water and nutrient applications because results are likely to be applicable to many fruiting trees, such as apples, cherries, pears, peaches and plums, all with main varieties depending on insect pollination. Almond trees are generally considered to be drought-tolerant , but growers are highly dependent on irrigation and nutrient inputs to produce high yields of top quality . Moreover, almond production in California is highly dependent on honeybee management to set a commercial crop, but declines in honeybee colonies and an increase in production acreage have resulted in honeybees becoming a limiting resource for almond in the USA .

Furthermore, California, as one of the largest irrigated agricultural areas in the world, is facing water shortages, and almond growers may be forced to reduce their annual water use . Almond was therefore selected to test experimentally the effect of pollination on fruit yield and foliage while taking into account the interactions of pollination, water and nutrient applications in a full-factorial design.Almond [Prunus dulcis D.A. Webb] is a subtropical obligate outcrossing tree that is intensively grown in large monocultures in California, where the climate is similar to its place of origin in the Middle East . Almond flowers must be pollinated by a compatible variety to produce a fruit . Bees, and possibly other insects, are the principal vectors for transferring the sticky pollen between flowers of different varieties, and there is little to no transfer by wind . The mature fruit consists of one or occasionally two kernels per fruit, surrounded by a shell nested inside a hull. The most popular variety for nut production in the USA is Nonpareil, which comprises around one-third of all marketable nuts produced in California . Compatible pollen donor varieties for Nonpareil include Padre, Mission, Wood Colony and Carrion. Almond trees flower from early February to mid-March in California, and fruits ripen from July to September, depending on variety, weather and age of the trees.The experiment was carried out from January to August 2008. The site was located in the Sacramento Valley, the northern portion of California’s agriculturally intensive Central Valley, in Colusa County, near the border with Yolo County . Colusa County produces around 5% of California’s marketable almonds. Precipitation in this area is low, with an annual amount of 268.7 mm . Rainfall occurs primarily from October through May, with a peak in January; no precipitation typically occurs from June to September. We selected young, productive trees for this experiment, because kernel quantity and quality of the whole tree can be measured, and accumulated resources are limited in smaller trees . The 3.2-ha study site consisted of Nonpareil trees that were grafted onto peach rootstock [Prunus persica Batsch] in 2005 and were planted in 2006 . The trees were approximately 1.75-m tall and had not been harvested prior to this study. The trees were planted 4-m apart within rows and 6.4 m between rows. Several ‘polleniser’ varieties compatible with Nonpareil were available in surrounding orchards located 100–300 m away from the experimental trees. These included a 16-year-old, 4.7- ha orchard with Mission and Carrion varieties and a 13-yearold orchard with Nonpareil and Wood Colony. The eight hives closest to the experimental trees were 300–350 m away and were part of the commercial pollination management system used by the grower. Padre pollen was placed every second day at 10:00 and 14:00 h at the nest entrance of the honeybee hives to maximise the amount of compatible pollen transported by honeybee workers to Nonpareil trees.

The total phenol content in aqueous samples was determined using the Folin–Ciocalteu assay. The samples were mixed with diluted Folin–Ciocalteu reagent and sodium carbonate and left react. The blue complex mixture was measured at 750 nm. With a calibration curve obtained using gallic acid, we determined the total phenolic content, expressed as milligram of gallic acid equivalents .The method reported in was used for the total flavonoid content determination. Using a suitable solvent to extract the sample, we obtained a flavonoid–AlCl3 complex. The absorbance of the complex was measured at a certain wavelength, and the flavonoid concentration was calculated using a standard curve. Quercetin was used as a standard, and the flavonoid content was expressed in terms of quercetin equivalent .The assay to determine 5-lipoxygenase activity involves measuring the conversion of arachidonic acid to leukotriene. This is typically achieved by incubating the enzyme with the substrate, arachidonic acid, and assessing the production of leukotriene products, often using techniques like high-performance liquid chromatography or enzyme immunoassays. The method described by Viji and Helen was used to assess the extracts’ effect on lipoxygenase.The phenolic aqueous extract was used to determine the percentage of α-amylase enzyme inhibitory activity. The α-amylase assay is a method for quantifying the activity of the enzyme α-amylase, which breaks down starch into simpler sugars. This assay typically involves mixing the enzyme with a starch substrate, letting it react, 25 liter pot and then measuring the conversion of starch to reducing sugars using colorimetric reagents, often with spectrophotometry

The α-glucosidase assay is a method used to measure the activity of the enzyme α-glucosidase, which catalyzes the hydrolysis of disaccharides into glucose. This assay involves incubating the enzyme with a substrate like p-nitrophenyl-α-D-glucopyranoside and then measuring the release of p-nitro phenol, typically using spectrophotometry, as an indicator of enzymatic activity. The phenolic aqueous extract was used to determine the percentage of α-glucosidase enzyme inhibitory activity.Wild-type D. melanogaster Harwich strain flies were reared using the method described by Oyeniran et al. in the Drosophila Laboratory unit of FUTA. This method indicates the temperature, humidity, and nutrition conditions to preserve the wild-type strain. For best health and reproduction, we used suitable containers with standard Drosophila food medium, controlling the population density and providing fresh medium on a regular basis.The choice of the eggplant flour concentration in the formulated food was based on a preliminary study on fly survival rate where various concentrations between 0.1 and 2% were used. Flies 3–5 days old were separated into 8 groups, with 40 flies per vial. The experimental groups and design are shown in Table 2. The incubator was set at 25 ± 1 ◦C. The fruit flies were sacrificed, and the obtained tissue homogenates were extracted. The tissue extracts were used to carry out in vivo assays for the determination of protein content, α-amylase activity, α-glucosidase activity, glucose level—using the Sigma Randox kit and expressing it in mg/dL—ROS level, MDA content, total thiol, CAT activity, GST activity, and SOD activity.As shown in Table 1 and Supplementary Figures S2 and S3, it was found that the ripe fruits contained considerable amounts of phenolic compounds such as phenol, cinnamic acid, quercetin, chlorogenic acid, and myricetin. All the polyphenols found in the ripe fruits were also found in the unripe fruits.

The ripe fruits had a total phenolic content of 2205.43 mg/100 g, while the unripe fruits contained 2354.94 mg/100 g of phenolic compounds. This suggests that the ripe fruits could be as useful as the unripe fruits and should not be discarded. This clearly shows how useful nutrients which could be utilized in food products’ development are wasted in farms due to lack of information.This study compared the effects of ripe and unripe fruit diets on diabetes in order to evaluate their biological potential; however, our main focus was on the effects of ripe fruits, which are not always reported in studies because ripe fruits are considered waste. Due to ripening, about 60% of indigenous eggplant Solanum anguivi lam fruits in Nigeria are left to waste in farms, because the ripe fruit is given little or no importance.Meanwhile, this unwanted fruit is rich in viable phytochemicals, as reviewed in [36], which explained the benefits of phyto-compounds such as hesperidin—known as a bioflavonoid with anti-inflammatory, skin-lightening, antimicrobial, and wound healing properties—and naringin—which was found to have broad pharmacological potential. In addition, myricetin was previously reported to have neuroprotective, analgesic, antihypertensive, antidiabetic. and hypolipemic potential. This clearly shows that many beneficial bio-active compounds from nature are wasted yearly due to the early ripening of fruits and vegetables as well as inadequate/poor storage facilities. This present study showed that the ripe fruit contains more vitamins and minerals than the unripe one, which are essential for the proper functioning of the body. Vitamins are essential nutrients that are needed in the body and are involved in cell regulation, body metabolism, growth, and development, physiological processes, such as iron absorption, and the immune response. We found that the levels of vitamins A, C, E, and carotenoids and of some minerals increased with ripening, which confirmed a previous report; the change in coloration of the Solanum anguivi lam fruit could be due to the synthesis of alpha-carotene and beta-carotene, precursors of vitamin A.

COX and 5-LOX metabolic products such as prostanoids and leukotrienes are implicated in degenerative diseases such as cancer progression and may represent important targets for cancer chemoprevention or treatment. From the in vitro results, the bio-active compounds present in the ripe eggplant extract could serve as dual inhibitors of inflammatory enzymes, able to block the COX and the 5-LOX metabolic pathways better than synthetic drugs . The ripe eggplant extrac bio-active compounds, as shown from the results, have either additive or synergistic effects on the two major enzymes in arachidonic acid metabolic pathways, thus inhibiting their inflammatory activities. Secondly, it could be suggested that eggplant extracts and related diet also possess another advantage, as they have small or no gastric toxicity, being natural products with a balancing effect compared with synthesized non-steroidal anti-inflammatory drugs such as sulindac and aspirin, which are commonly used but cause major side effects, such as ulcerative colitis, due to non-selective COX inhibition. This study clearly shows that the eggplant, especially if ripe, could display an enhanced anti-inflammatory potency based on polyphenol structure, which contains almost the same structural and functional groups as the above-mentioned drugs. This observation confirms the need to utilize natural products such as eggplant for non-selective dual COX/5-LOX inhibition without side effects. Some reports indicated that some plant products such as curcumin, tea, silymarin, resveratrol, and green and black tea, rich in polyphenols, are used as natural dual enzyme inhibitors, which means that eggplant extracts or and its related diets could exert beneficial, therapeutic effects through the modulation of COX and LOX metabolic pathways. In addition, it was also confirmed that chronic inflammation is implicated in the pathophysiology of diabetes, especially type-2 diabetes, and to that end, targeting inflammation may help ameliorate diabetes, preventing its sequence and other metabolic complications, which means that an eggplant-supplemented diet could exert multiple beneficial functions. Bio-active compounds such as vitamins, carotenoids, phenolic compounds, and flavonoids, which seem to be abundant in fruits, 25 liter plant pot whether ripe or unripe, could be the reason behind the low weight gain, the increased survival, and the decreased mortality rate of the flies. This study confirmed what was reported in [5], i.e., that diabetic fruit flies administered a high-sucrose diet show obesity-linked lipogenesis caused by the repartition of fatty acids toward esterification, away from fatty acid β-oxidation, possibly by increasing malonyl-CoA. Moreover, there is a relationship between movement and weight gain which is also linked to obesity, meaning that a healthy lifestyle involving exercise and nutritious food rich in phyto-compounds, minerals, vitamins, and fiber, and with a low GI could holistically enable body wellness, as suggested by Nwanna et al.. It was also observed that the ripe eggplant-inclusive diet was able to elicit positive effects better than the unripe fruit-inclusive diet, at the same time reducing the glucose,MDA, and thiol levels in the diabetic subjects compare to the untreated subject. There is connection between diabetes mellitus, oxidative stress, and reduced antioxidant activity, while the biomarkers of oxidative stress are related to reactive oxygen species, MDA, and thiols. Thiol is an organic compound containing a sulfur atom. It is also referred to as mercaptan.

Thiol acts as an antioxidant, stabilizing free radicals by accepting their unpaired electron. This means that a ripe eggplant diet, with its bio-active compounds, contains relatively more antioxidant molecules than an unripe eggplant diet. Furthermore, an increase in endogenous antioxidant enzyme activities was observed in the groups treated with the supplemented diet, and diabetes causes tissue injury in flies as a result of the increased production of ROS. The elevation in the activity of these enzymes might be a result of the scavenging ability of the bio-active compounds in the fruit diet, which eliminated the ROS species and exerted their effects by restoring the activity of antioxidative enzymes. SOD is an enzymatic antioxidant that defends against free radicals by converting superoxide anions to compounds with less damaging effects like hydrogen peroxide; it serves as the first-line defense mechanism against pro-oxidative injury in tissues, which is important for lifespan extension and survival of Drosophila melanogaster. CAT is an enzyme that converts lesser damaging compounds such as peroxides and H2O2 to water and molecular oxygen. CAT activity was reduced in the sucrose-treated and control groups; this could also be explained by the accumulation of H2O2. We found that the inclusive diet increased the activities of SOD and CAT, which suggests a quick response to fight free radicals by reducing the endogenous H2O2 that was generated and diminishing the toxic effects it could cause. GST is needed to prevent damage and serves as a defense mechanism. The ability of the Solanum anguivi lam fruits, whether ripe or unripe, to restore the damaged antioxidant enzymes status in sucrose-induced diabetic flies cannot be overemphasized. It is interesting that the fruit contains high levels of bioflavonoid compounds such as hesperdin, naringin, and myricetin, with high binding affinity for all the proteins of interest in this study; these compounds could serve as good inhibitors of the enzymes associated with diabetes and inflammation, although it was earlier reported that they have numerous biological properties such as anti-microbial, anti-lipidemic, anti-cancer, and antioxidant properties. Ripening increases the levels of some of these bio-active compounds, which further confirms the report of Nwanna et al. of the immerse benefit of the eggplant fruit especially, the wild species.Tephritid fruit flies are well-known agricultural pests, and there are approximately 4500 species worldwide . As typical herbivores, host plant expansion is an important survival strategy for tephritid flies, especially when introduced into new areas. Host plant expansion is the ability of an herbivore to use novel host plants without losing their ability to use their original hosts , which facilitates the establishment of tephritids when entering new geographic areas and expanding their damage . Therefore, understanding the mechanism of host plant expansion will be helpful for the control of tephritid pests. Host expansion is well documented in the most destructive species of the genera Anastrepha, Bactrocera, Ceratitis, Dacus, and Rhagoletis among tephritid flies because they have expanded their range worldwide . For example, the ancestral hosts of Zeugodacus cucurbitae  in India are primarily cucurbits, but it began to infest papaya in Hawaii , and it expanded its host range to include mango in Africa . The peach fruit fy Bactrocera zonata  expanded to oranges and tomatoes when introduced from southeastern Asia to Egypt . Because of the typical frugivorous pest, the tephritids spend some stages of life from eggs and larvae to pupae in the fruit of host plants. Therefore, the microenvironment of host fruits to which flies try to expand will have an important infuence on the survival and adaptation of fruit flies. Therefore, when tephritids successfully expand their host range from ancestral host fruits to new hosts, they must adapt well to the chemical and non-chemical properties of the microenvironment from the novel host fruits, including theirphytochemicals, color, and phenology. The color of the host fruit is an important cue to many fruit-infesting insects when selecting a new host .

The major organic acid associated with pulp total acidity is citrate, which begins to accumulate during stage II of fruit development, when the fruit and its juice vesicle cells enlarge rapidly . The accumulation continues for a few weeks, reaching a peak when the fruit volume is about 50% of its final value, then the acid declines gradually as the fruit matures. In most varieties, there is a slight increase in sugar content early in fruit development, but the major increase occurs during stage III, when the acid content declines . In citrus, the major translocated sugar is sucrose and in many varieties, it accumulates to double the level of glucose or fructose . Maturation index, which determines the fruit’s internal quality, is the ratio between total soluble solids and total acidity. As the acid content declines toward harvest, sugars account for most of the TSS. As already noted, climate plays a major role in fruit development and maturation. Most of the commercial citrus cultivars were selected or bred in the subtropical regions of the world, and they are therefore adapted to regions where maturation occurs during the cool season . In hot climates, such as in the tropics, fruit maturation is accelerated and the major factor affected by temperature is the fruit acid level, with a linear relationship between the accumulation of heat hours and acid decline . Therefore, in hot climates, the fruit reaches its maturation index faster than in colder climates, black plastic planting pots and tends to be too sweet. However, this is only part of the problem. Citrate catabolism is associated with an increase in alcohols, aldehydes and other secondary metabolites associated with reduced flavor and fruit decay .

Therefore, in hot climates, the time during which the fruit is harvestable and marketable is considerably shortened, and fruit decay occurs faster than in the colder regions . Hot climate has the opposite effect on color break, which requires the correct number of cold night-time to develop . Therefore, not only the fruit decay faster in hot climates, but their color does not fully develop, and in extreme cases may even remain green. One of the expected outcomes of climate change is warmer winter temperatures with shorter cold-night times . As most citrus cultivars are harvested during this season, the effect of global warming is expected to be negative on both internal and external citrus fruit quality.Sink strength is determined by the sink’s size and activity . In crop plants, it is defined in practice by yield parameters , and quality parameters, such as carbohydrate and protein levels. Fruit size is genetically controlled, but physiological parameters, such as sink position in relation to other sinks and source tissues, and the time it takes to develop, also affect sink size and therefore, its strength . In tomato, there are over 30 loci that define fruit size, with many genes acting to control cell division at various developmental stages . Practically, sink activity is defined as the rate of photo assimilate translocation and their contribution to growth and developmental processes relative to their accumulation. To simplify the discussion, we will refer here only to sugars, as the major photo assimilates in fruit in general, and in citrus fruit in particular. As with many other plant species, in citrus, sucrose is the major sugar translocated from the leaves to the fruit .

During fruit maturation, it is the major accumulated sugar, with a sucrose:glucose:fructose ratio of 2:1:1 in many cultivars and references therein. In many cases, sucrose accumulation is detected early in fruit development, indicating a higher translocation than utilization rate . As discussed further on, sucrose catabolism into hexoses within the fruit provides the central mechanism controlling sink activity, and therefore sink strength. Sucrose is hydrolyzed either to fructose and UDPglucose by sucrose synthase , a bidirectional enzyme, or to glucose and fructose by invertase, a unidirectional enzyme . Following hydrolysis, glucose and fructose are phosphorylated to glucose-6-phosphate and fructose-6-phosphate by hexose kinase and fructokinase, respectively, while UDP-glucose is phosphorylated to glucose- 1-phosphate by UDP-glucose phosphorylase. While SuSy is cytosolic, sucrose hydrolysis by invertase is performed in the apoplasm by cell-wall invertases, in the cytosol by neutral/ alkaline invertases, and in the vacuole by acidic invertases. The enzyme is modulated post-translationally by invertase inhibitor, which might act in vivo, but not necessarily in vitro . In a few plant systems, it has been shown that alteration of the activities of SuSy and various forms of invertase results in altered yield and/or carbohydrate levels, and thus altered sink strength. For example, one amino acid change in the tomato cell-wall invertase LIN5 enhanced specific activity of the enzyme, the rate of sucrose uptake and, overall, BRIX . Increased expression of cucumber SuSy induced sucrose and starch accumulation and increased fruit size .

Transgenic down regulation of tomato SuSy resulted in reduced sucrose uptake early in fruit development, reduced fruit set, reduced fruit number and reduced fruit size . Similarly, reduced expression of acid invertase and SuSy in muskmelon and cucumber, respectively, reduced fruit size and sucrose level . Phenotypes associated with reduced sink strength were also demonstrated in carrot roots by down regulating vacuolar and cell-wall invertases as well as SuSy . Taken together, these studies demonstrated the importance of invertases and SuSy for sink strength and supported the notion that sink strength is controlled, at least in part, within the sink cells and/or at translocation points, i.e., zones of phloem unloading.Sugar transport from the leaf to the collecting phloem is defined as sugar or phloem loading, and its release from the transport system, the releasing phloem, into the sink cell is defined as sugar or phloem unloading . Movement of photo assimilates from the leaves to the sink through the stem via the transport phloem is a complex process. Although a major driving force is the concentration gradient between source and sink according to the pressure flow hypothesis , long-distance movement requires in and out movement of solutes from the transport system to the surrounding tissue, temporal accumulation, and energy investment . The mechanism of sugar unloading has been investigated in a number of fruit and other sink organs, such as tomato, grapeberry, cucumber, apple, walnut and potato tuber, by microscopy, fluorescent dyes, immunolocalization of sugar transporters, and use of transporter inhibitors . Unloading is operated by two major mechanisms, symplasmic, in which sugar transport occurs through the plasmodesmata connecting the transport cells and the sink cells, and apoplasmic, where sink cells are not connected symplasmically to the transport cells, and transport must therefore cross membranes through the apoplasm, usually using facilitated transport mechanisms . In some fruit, such as cucumber, apple, and kiwifruit, unloading is apoplasmic throughout fruit development . In other cases, such as tomato fruit and grape berry, there is a shift between symplasmic and apoplasmic unloading , whereas in the potato tuber, the shift is from apoplasmic to symplasmic . In jujube, two shifts occur during fruit development; apoplasmic unloading early in fruit development shifts to symplasmic unloading, which then shifts back to apoplasmic unloading close to ripening . The two types of unloading occur simultaneously during walnut fruit and seed development, symplasmic in the seed and apoplasmic in the fruit . Overall, symplasmic unloading is considered faster than apoplasmic unloading . Therefore, the mechanism is dependent on the rates of sink development and photo assimilate utilization versus the rate and form of their accumulation, in order to avoid an increase in osmoactive molecules in the cytosol. For instance, drainage pot in tomato fruit and grape berry, fruit development and sugar utilization are rapid during the first half of their development, requiring symplasmic unloading. Later, when the fruit shifts from a utilizing to accumulating sink, the unloading rate is reduced by shifting it to apoplasmic. During the first half of potato tuber development, it accumulates soluble, osmoactive sugars, and therefore apoplasmic unloading is required, but during later stages, starch is accumulated, allowing a faster unloading. Jujube fruit is characterized by rapid growth during the middle stages of its development, and therefore apoplasmic unloading is interrupted by symplasmic unloading during this stage.As phloem unloading has never been investigated directly in citrus fruit, the mechanisms in the various fruit tissues are unclear . Nevertheless, potential mechanisms can be discussed based on the following: the kinetics of sugar transport into the various fruit tissues, vascular bundles, segment epidermis, stalk of the juice sac, and juice sac, as determined using photo assimilate distribution by 14CO2-feeding of leaves, through either continuous labeling or pulse-chase experiments , the steady state distribution of sucrose and hexoses, as well as the activities of sugar-metabolizing enzymes and their protein levels in the various fruit tissues, especially during intensive sugar uptake . While the activities of sugar-metabolizing enzymes have been well-studied and characterized, understanding their physiological role during the various stages of fruit development is more challenging.

In tomato, for instance, about 20 days postanthesis, SuSy activity decreased and the activity of an apoplasmic invertase, eventually identified as LIN5, was induced . This shift was associated with the well-studied shift from symplasmic to apoplasmic unloading and with the conversion of the fruit from utilizing to accumulating sink . It might be concluded, therefore, that in tomato fruit, SuSy activity is required to maintain a high rate of sucrose utilization, whereas invertase activity is associated with hexose accumulation. Clearly, the equivalent information is still missing in citrus fruit. In the following, photo assimilate movement and distribution, as well as the activities of sugar metabolizing enzymes and their protein levels are described for the various fruit tissues . In most of the studies, the activities of the various forms of invertases are defined by their pH optima and solubility. Herein, alkaline/neutral-soluble invertase is referred to as cytosolic invertase, acid-soluble invertase as vacuolar invertase, and acid-insoluble invertase as cell-wall invertase .The vascular bundle terminates near the segment epidermis. However, albedo cells are present between the bundle and the segment epidermis, and therefore phloem unloading is expected to occur primarily into albedo cells before sugar reaches the pulptissue . In young fruitlets and fruit, the albedo is the major tissue; cell division terminates within 4–5 weeks post-anthesis, and fruit growth during stage II is achieved by pulp expansion . Therefore, it might be assumed that most of the sugar in the albedo is transported, while only a minor part of it is required for albedo cell metabolism and development. Pulse-chase experiments demonstrated that most of the radiolabel remains in the post-phloem compartment, i.e., albedo cells, for about 24 h before reaching the pulp tissues, juice sac and segment epidermis . This slowing of sugar movement could indicate that most of the transport is via the apoplasmic path. The presence of insoluble acid invertase activity could provide an indication for this type of unloading; such activity has been detected in albedo cells, although at a lower level than in other tissues—the vascular bundle, segment epidermis, and juice sacs . In addition, the albedo contained considerable activities of SuSy, vacuolar invertase and cytosolic invertase ; in fact, the activity of vacuolar invertase was strongest in the albedo compared to other fruit tissues, indicating active storage of sucrose/hexoses in the albedo after unloading. Obviously, the presence of plasmodesmata and a symplasmic pathway between the vascular bundle and albedo cells cannot be ruled out at this stage.As already noted, the segment epidermis provides a continuous layer with the juice sac epidermis . It is considered part of the transport tissues, and therefore enzymatic activities are sometimes reported for the vascular bundle and segment epidermis together , although in other cases they are separated . A considerable percentage, about 30%, of the total radiolabel could be recovered in the epidermis, with maximal accumulation between 24 and 48 h after feeding in a pulse-chase experiment using grapefruit . With continuous labeling, about 50% of the total radiolabel was recovered in the segment epidermis within 24 h. However, when radiolabeled sugars were quantified in Satsuma mandarin after 48 h of feeding with 14CO2, the segment epidermis displayed the lowest amount per fresh weight or per fruit . These discrepancies could be due to different experimental designs or reflect cultivar differences. Regardless, the segment epidermis provides a strong sink, and movement of photo assimilates from this sink to the juice sac cells cannot be ruled out. Sucrose hydrolysis in the segment epidermis was mediated by relatively high activities of SuSy and soluble invertase .