Month: December 2024

We anticipate that the amount of potentially viable seeds found in CAwr debris due to granivory with various degrees of fruit wall coverage affect: seed dormancy and thus widen the range of germination in the seed bank from the cohort of seed that will fall all at once with the senesced plant and, availability to post-dispersal seed predation with opportunities for dispersal. Our results, combine with all previous evidence of the hybrids high genetic polymorphism, suitable life-history traits and higher fecundity both inside and outside its distribution range, provide a compelling argument for the hybrid to have displaced its progenitor lineages and for its capacity to be a successful invader .During the invasion process, the ability of a species to successfully persist and evolve depends on spreading its propagules . These offspring carry the resulting effects of biotic and abiotic selective forces including both natural and sexual selection. High reproductive rate characterizes successful invaders because it translates into thriving demographics allowing dispersal and range expansion . However, reproductive investment alone does not always explain invasiveness such as when natural selection has the potential to modify this over time . In multi-seeded fruits, understanding the effects of within-fruit seed characteristics is an opportunity to explore the effects of sexual selection. Sexual selection effects, procona buckets coupled with natural selection, may influence the success of an invasive lineage .

Seeds are the propagules of sexually reproduced plants and the majority of plant invaders rely on their dispersal . Most of the studies on seed or fruit traits in invasive plants either emphasize the effects of high fecundity or the rate of dispersal and life history traits . However other seed characteristics such as seed size , seed paternity and maternity and within-fruit seed position are variables that have been shown to influence seedling establishment, plant growth, adult plant size, ecology and reproductive output in noninvasive species . Intraspecific seed size variation is considered to be stable and tends to vary under density-dependent competitive situations . Variation in seed size also occurs within fruits and this is particularly apparent when the ovules are arranged in a linear arrangement within the fruits. In cucumber , the higher the fruit position on the mother plant and the within-fruit peduncular seeds were the slower to reach maturity and lowest dry weight . However, differences in fruit and ovule positions were compensated over time, when seeds had more time to mature inside the fruit . Seed size, from a broad phylogenetic to specific ecological interactions is a significant trait in most plant’s lifetime performance.Therefore, within-fruit seed size and other characteristics such as seed position and paternity, can have a relevant effect on invasive hybrid derived lineages. Therefore, I investigated how those within-fruit seed characteristics compare between a hybrid lineage relative to its progenitors. In particular, the present study compares the within-fruit seed characteristics and fitness among the invasive hybrid-derived California wild radish and its cultivated Raphanus sativus and wild R. raphanistrum progenitors in a noncompetitive setting. At the seeded portion, Raphanus fruits hold up to 13 seeds.

A relatively fine seed coat covers Raphanus seeds and its integral presence induces dormancy . Some of the features of the hybrid-derived CAwr reproductive biology may strongly influence its ability to adapt to new environments. The hybrid derived lineage fruits have multiple paternity and multiple lines of evidence suggest that the seed siring occurs in a non-random mating manner . Thus, the identity of the pollen donor is an important determinant in the sequence of the fertilization and seed placement within the pod-like fruit . Inside the fruit, seeds vary in size , with a general tendency to be heavier at the attached or peduncular end . The order of ovule fertilization in the Raphanus raphanistrum follows two patterns: ovules in stylar section first, followed by middle ones and lastly peduncular ones, or middle ovules first followed by stylar ones and peduncular ones. These patterns are explained by gamete selection at prezygotic mechanisms level as well as by the gynoecium internal structure . A central transmitting tissue structure, called septum, allows compatible pollen tubes to grow and by-pass ovules at stylar positions . However, I do not know if all three lineages have the same within-fruit seed characteristics. Our objectives are to compare seed weight among all three Raphanus lineages and their populations, to determine if within-fruit seed positioning influences seed weight, fecundity, and other morphological as well as fitness related characteristics, to compare if single and to mixed hand pollination crosses influence fitness values, to document the occurrence of multiple paternity in all three lineages, and to assess maternal and paternal effect on seed weight and other fitness related variables.Seed sources – The plants that produced the seeds used to breed the mother plants and the control fruits in these experiments were reared in a common garden during Spring 2005 and Winter 2006. The seed sources for those maternal and control plants are described in table 3.1.

The seeds are the result of natural open pollination in common gardens at the Agricultural Experiment Station at the University of California-Riverside . More details on how the plants were grown can be found in Ridley and Ellstrand . Control vs. mixed and single crosses – We measured seed characteristics from seeds extracted from: fruits obtained from open pollinated plants hereincalled control plants: at random I selected five mothers within each of three populations for each lineage and randomly chose three fruits for each mother plant for a total of 135 fruits, and from the offspring resulting hand pollinations from mix and single pollen hand pollination crosses performed in 2010. In total I performed 595 crosses, 336 mixed pollen crosses and 259 single pollen crosses, in a total of 83 plants: 23 CAwr, 30 Rr and 30 Rs, for more details on these crosses see “Paternity”. Viable seeds common garden – Fruit from control, mixed and single crosses were carefully opened with a cutting knife . Inside each opened fruit, seeds in all positions were examined. We counted the total number of seeds or seed set, including viable and unviable seeds as well as empty seed compartments. Seed viability was initially determined by visually inspecting the seed coat and by putting pressure on each seed between the thumb and the index fingers; when unviable, seeds had black and/or wrinkled seed coats and disintegrated easily. Before planting in soil, all “viable” seeds were soaked in 600-ppm giberellic acid solution and germinated in large trays lined with distilled water damped Whatman filters. The trays were placed in the dark of the laboratory and visited twice daily to record germination. After 5 days all seeds were transplanted in seedling trays with UC Soil mix III and moved into a temperature-controlled greenhouse. The greenhouse was visited daily to continue monitoring for germination or any other changes. After 53 days, the plants that survived were transplanted to common garden plots at the University of California-Riverside Agricultural Operations , where I monitored daily for floral buds emergence and first opening flower as well as any changes in the plant condition including signs of herbivory. The common garden design consisted of four plots of 11 m x 11 m at AgOps-UCR, where I planted the seedlings spaced by 1 m in all directions. The plants were watered once daily for 5 min with a sprinkler system until they all started to flower. Plants that survived to the end of the experiment were measured for total number of fruits produced and final plant height , procona florida container and collected to obtain final plant weight .Starting from the peduncular end , each viable seed was weighed to the 0.01 mg with an analytical balance .

The seeds were always extracted in the same order, and sequentially placed by columns in a labeled 96 well plate recording the row and column in a spreadsheet. Once all “viable” seeds of a fruit were placed in a plate, the section assigned to the fruit was securely taped and labeled with the lineage, population, mother plant and fruit ID’s to be stored for further analysis. This arrangement kept the seeds in order and prevented any translocations among positions and fruits. Lastly, the seeds were planted in a common garden to test fitness, always maintaining the same order of extraction from peduncular, middle, to stylar ends.Three within-fruit seed characteristics were tested: seed weight, within-fruit seed weight percentages, and relative withinfruit seed fecundity. Within-fruit seed weight percentages were calculated by dividing a given seed weight by the sum of all seed weights in a given fruit and multiplied by 100. Within-fruit relative fecundity was calculated by dividing the seed fecundity by the overall fruit fecundity where fecundity is defined by the total number of fruits produced. Within-fruit seed position was considered in two different ways: the seed positions per se recorded during the seed extraction process, and as seed position bins. To make the different fruit positions comparable, I divided fruit positions into three seed position bins corresponding to the peduncular, middle and stylar positions as described in figure 3.1. These seed position bins were based on our own observations and on the way I divided the fruits as I opened and removed the seeds. Within the seeded portion of the fruit, I divided the fruit in two. This central point was defined as the middle portion . Next, 2/3 away from that central point toward the basal part of the fruit I marked the peduncular portion . Then, 2/3 away from the same central point, this time towards the distal part of the fruit, I determined the stylar portion of the fruit. These variables included days to germination after planting, which constituted the time baseline for all other life cycle variables, such as, the days to floral bud emergence, and the days to first opened flower. Paternity – To assess the occurrence of multiple paternity on the three lineages I performed mix and single pollen crosses on mature unopened flower buds. Any mature flower bud with a corolla that was not tightly closed or that presented any opening as a result of damage was discarded. Immediately after performing hand pollinations , pollinated stigmata, fully covered with pollen, were covered with labeled tulle bags until the end of the experiment. The purposes of the tulle bags were to avoid unwanted pollen transfer, to protect the fruit from any predation and to allow proper maturation without losing the fruit once dried.At the beginning of May 2010, I collected the pollen sources plants for our hand pollinations from a natural well-established population of CAwr. The five already flowering plants were found in Hemet in the San Jacinto Valley in Riverside County, California. The chosen plants had different flower colors looked healthy, and had no pests or signs of herbivory. These plants were individually transplanted to gallon pots and then placed in a temperature-controlled greenhouse at UCR. In the greenhouse the five plants were watered daily, trimmed to prolong their flowering and maintained pest free until the end of the experiment. Mix pollen load crosses included different combinations of four pollen donors, and in one case three, out of the set of five described above. At a given time, the mixture of pollen donor plants depended on: 1) the outcome of initial single crosses that verified compatibility with the pollen receivers and also 2) pollen donor flower availability at a particular time. Single crosses were also used as a reference through the microsatellite analyses. Once a mother plant was pollinated with a particular pollen mix, the same mix was used to pollinate the rest of the flowers on that same mother plant. Despite evidence for lack of effect in the amount of pollen on reproductive output , I equalized amounts of pollen applied per donor across crosses as follows . For single crosses, I always removed the pollen from a single new flower by gently tapping all six anthers against the bottom of a clean Petri dish. For mixed crosses, I selected two anthers per father, totaling eight anthers for all four fathers. All eight anthers were dissected from new flowers with tweezers and tapped against the bottom of a clean Petri dish. The pollen collected from all anthers was blended by gently swirling the pollen with a clean piece of folded lab tissue held with tweezers.

Fresh weight was used due to the large number of plants and measurements being done in situ in the field setting. All measurements were made in kg. To measure the BRIX value of the fruit, the collected tomatoes was taken to the lab where the juice was squeezed out and measured on a refractometer . The yield and BRIX for each plant were multiplied together to get the BRIX x Yield Index , which gives an overall fruit quality measure, accounting for variations and extreme values in either measurement. It should be noted that while BRIX is used as a standard quality measure, BY is a composite value that folds in yield to assess kilograms of soluble solids per plant and is used to measure commercial quality and not consumer quality .Leaf shape and complexity measures were performed for the 2014, 2015, and 2016 field season. Subsequently, primary leaflets were used for imaging and analysis of shape and size as described , and the images then processed in ImageJ . The images were cropped to individual leaflets maintaining the exact pixel ratio of the original image, and then cropped again to only include the single leaflet using a custom Java script written for FIJI . Single leaflet images converted to a binary image as black on a white background and smoothed to allow for the exclusion of any particulates in the image and were then processed in SHAPE , plastic plant pot a shape analysis software.

Leaflet images were imported and then aligned along their axes so that all images faced the same direction and then were processed using elliptical Fourier analysis based on the calculated number of harmonics. PCA was performed on the resulting eFourier analysis and the Principal Components used for subsequent analysis . The PC values were used for all subsequent leaflet shape analyses. Total leaf area for each plant was measured by imaging the whole plant and a 4 cm2 red square and then processed in the Easy Leaf Area software .Plants were grown in the field to flowering stage for the lines M82, BIL 260, and sub IL 4-3-4. Three leaf punches from three separate leaves were collected from each plant every two hours for twenty-four hours. For sugar analysis leaf punches were then heated in 500 mL of 100% Ethanol for 20 minutes at 80 C and the liquid collected and stored at -80 C. The discs were then heated in 100% ethanol at 80 C for 20 minutes a second time. The discs were then placed in 500 L of 5% Sodium Hydroxide and heated for 20 minutes at 80 C. After cooling to root temperature 125 L of 5M HCl was added to neutralize the solution and then the supernatant discarded. The discs were then resuspended in 500 uL of 50 mM Sodium Acetate Buffer and ground by hand 2 mL Eppendorf tubes , after which they were spun down at 13,000 rpm for 1 minute. 25 L of starch degradation mix, consisting of Amyloglucosidase and a-Amylase in 50 mM Sodium Acetate Buffer, was added to the solution. The solution was incubated at 65 C for 24 hours and then the enzymes inactivated by heating at 80 C for five minutes. To measure sugar and starch content of the leaves, the solution was processed by the sugar assay as described by Rowland et al., 2019.Three to five leaf punches per plant were collected from individual leaves for each genotype.

The leaf punches were cleared by heating in 100% EtOH for 20 minutes three consecutive times and then heated at 80 C for 20 minutes in 5% NaOH and then the liquid discarded. After this 50% Bleach was added to the discs and they were incubated at root temperature for 20 minutesand then rinsed several times with water. The leaf discs were stained with 1% safranin and then imaged on an Eclipse C1 plus microscope at a fixed magnification .Count data of the DEGs was normalized across all samples prior to UMAP dimensionality reduction. UMAP dimensionality reduction was performed using the Julia package, UMAP. In order to identify smaller sets of genes with similar expression patterns, Mean Shift clustering was performed . GO enrichment analysis using the R package, GOseq , was performed on the 39 generated clusters. Cluster 19 was found to have GO terms “extracellular region” and “carbohydrate metabolic process”, among others. WGCNA was used to construct gene coexpression networks using normalized expression data of genes from cluster 19. Pearson correlation cutoff value of 0.95 was used. Separate networks for BIL260 and M82 were constructed. Networks were visualized using Cytoscape .BIL260 x M82 back crosses were made by removing the stamens from the female plant, and then these flowers were painted with pollen from the “male” plant with a paint brush. Flowers were bagged to tag and isolate from other nearby flowers and collected when mature. F1 seed were grown up in the greenhouse to generate F2 seed. The F2 plants were then grown in a random order in the field to perform analysis and collect seed. These plants were labeled 1 through 400. The F3 plants were grown in the greenhouse to characterize the plants for leaf shape, vasculature, and BY.The fleshy fruits produced by strawberry , tomato , and many other horticulturally important plants are susceptible to post harvest decay by gray mold, a devastating disease caused by the necrotrophic fungal pathogen Botrytis cinerea . Botrytis cinerea can infect most organs of the plant but is especially destructive on ripe fruit and senescent tissues of dicotyledonous hosts .

Gray mold renders strawberries unmarketable and often causes significant postharvest losses under conditions favorable for pathogen growth . The mechanisms of defense against B. cinerea are physiologically and genetically complex and markedly differ from the gene-for-gene resistance and programmed cell death mechanisms commonly triggered by biotrophic pathogens . As with other necrotrophic pathogens, B. cinerea pathogenesis is promoted by fruit ripening and host cell death . Consequently, genetic variation for resistance to gray mold tends to be subtle, limited, and quantitative, which undoubtedly underlies the paucity of studies on breeding for resistance to this pathogen . Because natural genetic resistance has been insufficient to prevent post harvest gray mold disease development, preharvest fungicides are often applied to suppress pathogen growth and minimize post harvest losses . Controlling B. cinerea with fungicides is difficult because the airborne inoculum is present year round, the host–pathogen interactions are complicated, and the pathogen rapidly evolves resistance to fungicides, particularly after repeated applications of specific chemicals . Moreover, preharvest foliar applications of fungicides have not been shown to be effective for reducing post harvest gray mold incidence in strawberry fruit possibly because many fruit infections arise from contaminated flower tissues . The development of gray mold resistant cultivars has been challenging in strawberry and other hosts because most genotypes are highly susceptible, strong sources of natural genetic resistance have not been identified, plastic planter pot and resistance mechanisms are quantitative . The feasibility of selecting for increased resistance to gray mold has not been deeply explored in strawberry, a species where limited studies have been undertaken to shed light on the genetics of resistance and assess genetic variation for resistance . The problem of breeding for resistance to gray mold has been most extensively studied in tomato, albeit without achieving robust or foolproof solutions . Genetic studies in tomato and Arabidopsis leaves have identified multiple small-effect quantitative trait loci that only account for a small fraction of the genetic variation for resistance, seldom translate across genetic backgrounds, and have not solved the problem of breeding for resistance to gray mold . Although genetic studies of similar depth and breadth have not been undertaken in strawberry, previous studies have not uncovered strong sources of resistance to gray mold . We suspected that selection for increased fruit firmness and other fruit quality traits that extend shelf life pleiotropically increased resistance to gray mold in strawberry. While hypotheses can be formulated from insights gained from genetic studies in tomato and other hosts , natural genetic resistance appears to be negligible and quantitative and additive genetic correlations between gray mold resistance and fruit quality phenotypes are unknown in strawberry . The susceptibility of strawberry fruit to B. cinerea increases during ripening , which suggests that susceptibility factors accumulate independent of defense mechanisms during fruit maturation and senescence, as is typical for this necrotroph . Changes in fruit firmness and other fruit quality traits associated with fruit maturation and ripening in tomato have been shown to increase susceptibility to B. cinerea . Although previous studies have been somewhat inconclusive in strawberry, firm-fruited cultivars are predicted to be more resistant to B. cinerea than soft fruited cultivars . Moreover, ripening induced differences in proanthocyanidin and anthocyanin accumulation have been predicted to affect B. cinerea resistance in tomato and strawberry .

To more deeply explore the genetics of resistance to gray mold and assess the feasibility of applying genomic selection for increased resistance to gray mold in strawberry, we developed and studied training populations segregating for fruit quality traits predicted to affect shelf life. Genomic prediction approaches are particularly attractive for post harvest traits that are difficult and costly to phenotype in strawberry but still require sufficient accuracy to complement phenotypic selection and achieve genetic gains . The training populations for our studies were developed from crosses between firm-fruited long shelf life cultivars and soft-fruited short shelf life cultivars. Although the gray mold resistance phenotypes of the parents of these populations were unknown, our hypothesis was that selection for extended shelf life has pleiotropically increased resistance to gray mold in strawberry, primarily because fruit of LSL cultivars deteriorate more slowly in post harvest storage than those of SSL cultivars. We describe a highly repeatable artificial inoculation protocol for gray mold resistance phenotyping developed for the GS studies described herein. Finally, we discuss the prospects for increasing genetic gains for resistance to gray mold through the application of genomic prediction approaches.We developed a high-throughput protocol for post harvest phenotyping of B. cinerea disease progression and symptom development on ripe fruit. Spore suspensions of the B. cinerea strain B05.10 were produced from spores grown on potato dextrose agar as described by Petrasch et al. . Uniformly ripe fruit were harvested at sunrise, avoiding fruit that were under- or over-ripe. The fruit were immediately transferred to cold storage and inoculated the day of harvest. Several incubation temperatures were tested to identify the optimum temperature for B. cinerea growth and development with a minimum of contamination from other post harvest decay pathogens. Fruit were placed on 30-cell plastic egg hatching trays with dimensions of 29 cm 29 cm and 4.5 cm  4.5 cm cells. The fruit were punctured once near the center with a 3 mm sterile pipette tip to an approximate depth of 1–2 mm. Ten ll of the B. cinerea conidia suspension was placed on the surface of the puncture. The inoculated fruit were incubated in a growth chamber at 10 C and 95% humidity for 14 days. Disease symptoms were assessed daily after inoculation by manually measuring lesion diameter and determining the number of days until external mycelium was evident on the surface of the fruit near the wound site. Fruit were phenotyped until mycelia covered the entire surface of the fruit. Spoiled fruit with infections outside of the inoculation site or caused by decay organisms other than B. cinerea were removed from the experiment. Genome-wide association study , QTL mapping, and GS analyses were applied to LD at 8 days post inoculation and EM.Fruit quality phenotypes were measured on one to four fruit harvested from individuals in the multifamily population at harvest. The fruit were photographed with a Sony a 6000 camera equipped with an E PZ 16–50 mm F3.5–5.6 OSS lens . Photographs were processed with a custom macro in Fiji to obtain RGB color metrics . RGB colors were subsequently converted into Lab colors using the convert Color function in R . Fruit firmness and fruit diameter were assessed on whole fruit using a TA.XT plus Texture Analyzer with a TA-53 3 mm puncture probe . Fruit samples were frozen at 20 C in WhirlPakVR Homogenizer Blender Filter Bags for quantifying titrable acidity , SSC , and total anthocyanin concentration .

In apple fruit, sucrose as well as sorbitol enters the parenchyma cells via the apoplastic pathway after being released from SE-CC complex. In many species that employ apoplastic unloading for sucrose in sink cells, sucrose is mainly converted to glucose and fructose by CWINV in the cell wall space and then transported into the parenchyma cells by hexose transporters. CWINV is typically considered as a sink-specific enzyme and its activity is usually very low in source leaves. However, we found that, except for MdCWINV3 in 40- DAB fruit, the expression of MdCWINVs was much lower in the fruit than in the shoot tips where sucrose unloading is symplastic. In yeast cells expressing apple SOTs, sorbitol uptake is competitively inhibited by glucose and fructose but not by sucrose. So we postulate that most sucrose is directly transported into the parenchyma cells by plasma membrane-bound SUCs in apple fruit to avoid inhibition of sorbitol uptake by sucrose-derived glucose and fructose. Increased sucrose import into transgenic fruit did not alter the activity of CWINV but significantly elevated the transcript levels of both MdSUC1 and MdSUC4 , indicating that more sucrose is taken up into the parenchyma cells in the transgenic fruit. There are two pathways for sucrose breakdown in the cytosol of fruit parenchyma cells: conversion to fructose and glucose by NINV or to fructose and UDP-glucose by SUSY . The upregulation of transcript levels of MdNINV1, MdNINV3, and MdSUSY1-3 and activities of NINV and SUSY in the transgenic fruit , which is indicative of higher availability of sucrose in the cytosol, generates more fructose. This, combined with a lower FK2 transcript level and a lower FK activity , plastic pots large makes enough fructose available in the cytosol for accumulation in the vacuole of the transgenic fruit to largely compensate for the reduced level of sorbitolderived fructose.

The higher NINV activity is also expected to elevate the glucose level in the cytosol, which may have led to higher transcript levels of MdHKs and a higher HK activity through glucose signaling and a higher dark respiration rate in the transgenic fruit . The higher HK activity detected in the transgenic fruit is similar to that of rice leaves in response to glucose manipulation. However, we found that increases in both HK activity and the glucose concentration did not enhance, but rather diminished, the accumulation of G6P in the transgenic fruit. This is likely due to a decrease in F6P flux from phosphorylation of fructose along with an increase in dark respiration such that more G6P was reversibly converted to F6P. Our result is consistent with the finding that glucose derived from sucrose contributes to the hexose phosphate pool more than fructose derived from sorbitol or sucrose in the apple fruit. Despite a higher glucose flux going through dark respiration in the transgenic fruit, more glucose is still available for transport into vacuole for accumulation as indicated by the 3–6-fold increase in glucose concentration in the transgenic fruit at harvest. Higher fruit glucose levels have also been reported for these anti-sense plants by Teo et al. but to a lesser degree. It is interesting that greater import of sucrose did not significantly increase its concentration in transgenic fruit except at 74 DAB. We think that two factors may have contributed to this near homeostasis of sucrose in the transgenic fruit. First, upregulation of sucrose breakdown described above uses more sucrose. Second, downregulation of MdSPS3 and MdSPS6 transcript levels and SPS activity in the transgenic fruit makes less sucrose re-synthesized from F6P and UDP-glucose.

While upregulation of SUSY in response to increased sucrose supply was observed in both fruit and shoot tips of the transgenic plants, NINV responded in the transgenic fruit but not in the shoot tips. The exact reason for this difference is not known, but differences in sucrose concentration and/or presence of different isoforms of NINV between fruit parenchyma cells and shoot tips might exist. Our findings on activities of SUSY, NINV, FK, HK, and SPS are not in agreement with those reported by Teo et al.. We believe that the discrepancy might be related to the difference in the way fruit samples were taken. In our study, it took about 2 min to cut and freeze fruit samples on site in the orchard, but in Teo et al.all harvested fruits were placed on ice before being transported to the laboratory and it was only after several quality indices were measured that the cortical tissues were frozen for further analysis. Because import of sorbitol and sucrose into fruit stops upon detachment from the tree, both enzyme activity and gene expression may be altered if they are not frozen in liquid nitrogen in a very short period of time. In addition, strict cropload CK in our study as reflected in much larger fruit might have made the difference between the transgenic fruit and the CK easier to be detected. Most of the hexoses and sucrose in fruit parenchyma cells are stored in the central vacuole that occupies >80% of the cell volume. Transcript levels of MdvGT1 and MdvGT2, both of which are vacuolar glucose transporters encoded by two Malus orthologs of AtvGT, were higher in the transgenic fruit than in the CK , suggesting that more glucose is transported into the vacuole of the transgenic fruit. This is consistent with the glucose concentration measured on bulk fruit samples .

In addition, tonoplast monosaccharide transporters can transport both glucose and fructose into the vacuoles, and Arabidopsis TMT1 activity for fructose is approximately 30% of that for glucose. In the five Malus orthologs of TMT, it is possible that proteins encoded by MdTMT1 and/or MdTMT2 have high ability to transport fructose, and the enhanced expression by MdTMT1 in the transgenic fruit might indicate a regulatory response to the reduced flux of fructose derived from sorbitol. Alternatively, as fructose-specific TMTs have not been identified in fructose-accumulating fleshy fruits, the upregulation of MdTMT1 could be triggered by higher levels of glucose derived from sucrose in the transgenic fruit. In addition to hexoses, the vacuoles in ripening apple fruit accumulate a high concentration of sucrose. So far, no SUC has been identified to have proton-coupled anti-port activity for loading sucrose into the vacuole, but AtTMT1/2 probably represents a proton-coupled anti-porter capable of transporting both glucose and sucrose into the vacuole. A recent report on TMTs in sugar beet indicates that one of the two TMT2 proteins has developed specific affinity to sucrose and is responsible for sucrose accumulation in the taproots. The expression patterns of both MdTMT1 and MdTMT2 are in general agreement with that of sucrose accumulation in our apple fruit. It has been demonstrated that interruption of carbohydrate import into fruit by girdling or adjustment of cropload did not alter the fructose level in apple fruit. Contrasting light exposure did not appear to affect peel fructose level either. The data obtained from the transgenic fruit in this study provides further evidence for supporting the idea that the Sucrose cycle and the associated transport system operates to maintain the homeostasis of fructose in the apple fruit. From an evolutionary perspective, having fructose homeostasis in the apple fruit may help seed dispersal for this species because fructose is the sweetest among all the soluble sugars present in fleshy fruits. In conclusion, when sorbitol synthesis is decreased by anti-sense suppression of A6PR in the source leaves of apple trees, less sorbitol but more sucrose is transported from the leaves to the fruit. In response to the lower sorbitol/higher sucrose supply, sorbitol metabolism is downregulated, whereas breakdown of sucrose is upregulated in the transgenic fruit to compensate for the decreased flux of fructose derived from sorbitol. This altered sugar metabolism, together with corresponding changes in the sugar transport system, black plastic nursery pots leads to near homeostasis of fructose and sucrose and much higher levels of glucose and galactose in the transgenic fruit. This study clearly demonstrates the metabolic flexibility and the advantages of having two transport carbohydrates in sorbitol-synthesizing Rosaceae tree fruit species and the central role of the Sucrose cycle and the sugar transport system in determining sugar metabolism and accumulation in fleshy fruits.The global fruit market has grown appreciably in the past decade. The absence of seeds from fruit that is consumed has become one of the most appreciated traits by consumers. Reducing seed content without changing the size of the fruit is one of the main objectives of the cultivation of many fruit trees. The goal is to enhance the experience of fruit consumption by consumers and improve the quality of fruits for food processing . Sugar apple  is a tropical fruit species in the family Annonaceae. Seedless fruits have been described for some spontaneous mutants of A. squamosa whose origin is still unknown .

They include the Cuban cultivar Cuban seedless , with good fruit characteristics but lower productivity than fertile cultivars ; Brazilian seedless originally identified in northeast Brazil, which produces small, asymmetric fruits that frequently perish ; the Thai seedless mutant that produces normal size fruits among other types with apparently similar fruits, such as in the Philippines and Hawaii . Lora et al. were among the first researchers to examine details of the seedless trait in A. squamosa in Ts. This variety produces fruit following pollination and fertilization. The authors demonstrated that seedlessness results from a deffect in ovule development where the outer of the two integuments sheathing the nucellus fails to form. This deffect directly mirrors the effect of INNER NO OUTER loss of function mutants in Arabidopsis thaliana . INO encodes a putative transcription factor belonging to the YABBY family. Members of this family are involved in the determination of abaxial identity in a variety of plant organs . Lora et al. isolated an A. squamosa INO ortholog and demonstrated the association of the Ts mutant with an apparent deletion of the INO gene, indicating a candidate gene for the seedless trait. The Bs variety was also evaluated in a breeding program undertaken at the State University of Montes Claros. Results showed that the absence of seeds was also associated with failure in the development of the external integument of the ovule and in the development of seeds, similar to that observed in Ts . In addition, preliminary studies of inheritance of the stenospermocarpic absence of seeds involving F1 progenies in Bs also indicated the same expected recessive nature for the mutation as in Ts . However, it was not known whether the molecular basis of the INO deletion described is widespread in A. squamosa and may be responsible for the case of aspermia described in Bs. In Arabidopsis ino mutants, the absence of the outer integument leads to failure in development of the embryo sac so that the ovules do not attract pollen tubes and degenerate . In contrast, Lora et al. found that the embryo sac fully formed in the Ts line, and was successfully fertilized, but this fertilization did not lead to the formation of seed. Santos et al. went further in analysis of Bs and showed that 72 h after pollination the embryos formed, but after seven days a degeneration of embryos and endosperm was observed, forming the sterile aborted seed with a whitish color and smooth consistency. It has been hypothesized that the more robust ovules of A. squamosa, with a much thicker inner integument than is found in Arabidopsis, can better support the development of the embryo sac in the absence of the outer integument and that this is the reason for the further embryo sac development in A. squamosa . The resulting initial stage of seed formation triggers the initiation of fruit development, explaining the stenospermocarpy. The present study addressed the following questions: Is the inheritance of the presence/absence of seeds in A. squamosa monogenic?; What are the molecular details of the INO gene deletion?; Is there a complete correspondence of homozygosity for the deletion with the seedless phenotype; What is the relationship between the known seedless accessions, and Can codominant molecular markers specific to INO be designed for use in assisted selection?Wild-types M1, M2 and M3 and seedless Bs lines were previously described by de Souza et al. , and Ts was described by Lora et al. .

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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