Plants employ a large array of strategies, both direct and indirect, to minimize the effect of seed predators. These include alternative strategies of regeneration, seed banks, abundant seed crops, altering phenology, spatial and temporal variation in seed productions and increasing plant defenses . It has also been demonstrated that pre-dispersal predation can facilitate the stratification of the seeds facilitating germination . A hybrid-derived lineage that has already successfully invaded a new habitat represents a useful model because, when combined with its progenitors, it allows for a replicated study of the invasion process. Here, I focus on the hybrid-derived California wild radish, which is an invasive lineage in western North America . Its progenitors, the cultivated radish Raphanus sativus and the wild radish R. raphanistrum, were introduced in western United States around mid 1800 . These two lineages naturally hybridize, and there is evidence for the hybridderived lineage to have originated from interspecific bidirectional hybridization between them . Genetically based differences between both progenitor lineages and a partial and temporary reproductive isolation during the first generations of hybrids results from a single reciprocal translocation . The polymorphism in fruit, flower color and shape, root morphology, chemical and structural defenses found in Raphanus lineages has been the focus of numerous studies in ecology, evolution, genetics and agricultural and food chemistry . Anecdotal observations have reported that bird pre-dispersal predation of seeds can be extensive in all three Raphanus lineages and birds are arguably the primary consumers of pre-dispersed seeds over invertebrate granivory . When granivory is excluded, the hybrid-derived California wild radish exhibits superior fitness compared to its parental lineages in common garden experiments across its Californian distribution . However, black plastic plant pots wholesale the hybrid’s relative fitness and that of one of the progenitors in the presence and absence of granivores, is unknown.

Our aim was to answer the following questions: what species of bird granivore is the main consumer of radish seeds? how much does the bird granivore affect relative fitness and relative potential fitness? are the variables likely to affect the birds’ selection of individual plants such as days to germination, plant final weight, total fruit production and potential reproduction, correlated with fruit damage? and are there viable seeds in the debris due to granivory resulting from the bird foraging behavior under damaged plants? In addition, the comparison among lineages allowed us to better understand novel biotic interactions in a successful invasive hybrid-derived lineage and to propose a mechanism that led to the replacement of both progenitors .Seed sources – The seeds used to breed the mother plants in the present study came from plants reared in a common garden during Spring 2005 and Winter 2006. The seed sources for the first generation of maternal plants are described in table 2.1. The second generation seeds are the result of natural open pollination in common gardens at the Agricultural Operations fields at the University of California-Riverside . More details on how the first generation plants were grown can be found in Ridley and Ellstrand . Common garden and experiment design – The common garden experiment took place during Spring and Summer seasons of 2010 at AgOps-UCR. Three replicate sites, each one consisting of two plots of 7 m by 7 m, were planted with 36 plants placed in a 6 x 6 grid with 1 m spacing in rows and columns. One of the plots at each of the three sites was covered with 3/4″ x 3/4″ orchard mesh to exclude above ground vertebrate damage while the other plot remained unprotected. These two conditions created two different treatments for the plants to grow in: protected from vertebrate seed predators and unprotected to vertebrate seed predators. In both cases the plants were exposed to open pollination, invertebrates and potentially underground vertebrates. All plots were oriented in the same North-South direction. We selected at random 8 seeds from 4 different mothers within each of the 9 above-mentioned populations for a total of 288 seeds. These seeds were divided in groups of 36, such that all mothers were represented in those 8 groups by 2 seeds. That is, each population had 4 seeds, for a total of 12 seeds per lineage.

These 8 groups of seeds were germinated in Petri dishes at the beginning of March and transplanted into seed starting trays filled with sterilized UC Soil Mix III at a climate controlled greenhouse. Once the seedlings had attained a three-leaf stage, 6 of the 36- grouped seeds were transplanted to the pre-water and plowed field plots. The two additional groups of seeds were used to replace any seed that did not germinate or any seedlings that did not survive the transplanting process. The plants were watered once daily for 10 min with a sprinkler system until most of the plants had started to flower. To maintain favorable abiotic conditions for the plants that flowered later, the watering persisted only every-other-day for 5 min. Granivores – We visited the sites at AgOps at least every two days to ensure that the experimental conditions were kept consistent during the entire length of the study. During those visits I also spent time observing the foraging behavior of the birds that began when fruits had attained a fully formed size. Once I became familiar with the birds foraging patterns, I spend an afternoon filming their behavior. Videos were captured with a digital video camera on a tripod. Videos are available as supplemental information.Fruit damage, fecundity, fitness related values and debris due to granivory – Variables related to morphology, damage and fitness were recorded before planting, during the experiment, and after the surviving plants were collected. All seeds were weighed to within 0.01 mg with an analytical balance . The germination and growth of seeds in the dish was recorded daily. At the end of the experiment when the plants were dry and had senesced, I recorded the final plant weight to within 0.001 g. To calculate fecundity and fruit damage, I counted total number of: damaged fruits that included all fruits with clear signs of missing or damaged sections, whole dropped fruits that were found detached from the dry plant, and whole attached fruits. With these variables I calculated fruit damage and fecundity. We counted total numbers of: flower buds, flowers, whole empty pedicels , and broken or pedicel scars on the stems. We also collected the fruit material or debris accumulated under heavily damaged plants, herein referred to as “debris due to granivory”, to discern what was discarded during the birds foraging behavior.

Potential seed viability was 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 crumbled easily. All the previously described values and those in table 2 allow us to calculate relative fecundity and relative potential fecundity of plants in unprotected and protected plots. Because I did not count number of seed per fruits, I calculated the number of seeds based on the average number of seeds per fruit per populations. These average values,listed in table 2, were obtained from a previous study where I counted total number of seeds from 884 fruits that belonged to the same populations represented here . We consider these values appropriate to extrapolate the number of seed in our study because: the plants that produced them developed from pure lineage seeds from the same populations represented in our study listed in table 2.1, the plants were grown under similar conditions to the present study, and the plants were exposed to open pollination . Total number of seeds were extrapolated for a given plant by: multiplying total number of whole fruits per plant by the average value of seeds in table 2.2 according to the population of origin, black plastic plant pots bulk followed by multiplying total number of damaged fruits by the 2/3 of the average number of seeds according to the population of origin in table 2.2, and finally by adding the numbers obtained for whole and damaged fruits. Fecundity and female fitness values were calculated as follows. The average number of extrapolated seeds per population was calculated by dividing the total number of extrapolated seeds divided by the total number of fruits per population. Relative fecundity is the average number of extrapolated seeds divided by the highest average number of extrapolated seeds. Potential reproduction was calculated by adding flower buds, flowers, whole empty pedicels broken to whole, damaged and dropped fruits for a given plant. The average potential reproduction was calculated by adding the potential reproduction for a given population or lineage and dividing by the total number of plants and multiplied by 100. Finally, the percentage of the relative potential fecundity was calculated by dividing a given average potential reproduction to the highest average one among for populations and lineages separately and then multiplying by 100. Our fitness values did not explicitly include male fitness. Nevertheless I know based on prior studies in plants of the Raphanus lineage that male fertility is highly influenced by environmental factors and weakly correlated with female fertility values . Data analysis – Data were normalized as needed either with log-normal or Box Cox transformations using functions in R . Significant P values were adjusted a posteriori with sequential Bonferroni test to adjust for type I error . We used one-way analysis of variance to tests the effects of treatments and lineages on total fruit production. Variables related with fruit damage and with fitness were compared in pairs among lineages and between treatments with Wilcoxon tests. The effect of the treatments on relative fecundity and relative potential fecundity as well as average number of fruits and seeds were tested for significance with Fisher exact tests. These tests were performed to individually compare CAwr values to its progenitors. We also compared fecundity values to the highest ones with chi-square tests. Total number of fruit damaged was correlated using Spearman correlation coefficients and covariance to variables possibly related to final fruit production and general performance. Those variables included: days to germination, final plant weight as well as total number of fruits and total potential reproduction. In this case each lineage was tested independently.Flower buds, flowers and pedicels – No differences were found between lineages and treatments in average number of flower buds and open flowers . With respect to the average number of pedicels, values for CAwr from protected and unprotected plots are significantly different and higher than both progenitors under protected treatment as well as for the cultivar under unprotected treatment, respectively . Fruits with and with no damage – We only found damaged fruits in plants that were collected in unprotected plots . Consistent with our previous results, average numbers of fruits with damage are significantly different among lineages and treatments as revealed by Wilcoxon tests . Whole undamaged fruits were categorized as either attached to the dry plant or detached and on the ground. The cultivar Rs differs significantly from CAwr and Rr on lower average number of whole dropped fruits, whereas both wild lineages, CAwr and Rr, are comparable . No differences are found in the average number of whole attached fruits among lineages with the exception of CAwr and Rs from protected plots . The average proportions of damage, calculated as total number of damaged fruits over the total fruits produced for each lineage and population, are listed in Table 2.3. When the damage is estimated based on seeds removed, calculations of damage per population are reduced by at least 33 % and at most by 60% relative to the damage calculated based on fruit damaged. Damage, based on seeds removed, was calculated as the total number of seed removed divided by total number of seeds produced. As mentioned earlier, I did not count the total number of seeds per fruits during this experiment. Fruits from Cst-CAwr suffer higher damage than interior populations . Fruit production – Total fruit production does not differ under protected or unprotected treatments but does differ among lineages . The cultivated Rs lineage produced fewer fruits in protected treatments relative to both wild lineages, significantly differing from both CAwr and Rr . However, under unprotected conditions, Rs only substantially differs from unprotected and protected CAwr fruit production . Fecundity and fitness related values – CAwr has significantly higher fecundity in protected plots than in unprotected ones, as shown in table 2.4.

Glucose-1-phosphate adenylyltransferase was highly down-regulated in symptomatic fruits while invertase was clearly up-regulated in the peel of apparently healthy and symptomatic fruits from the infected orchard. Sucrose symporter was up-regulated in infected, symptomatic fruits. The terpenoid pathway was affected by HLB disease, as shown by the down-regulation of terpene synthase cyclase . Induction of lipid transfer protein was observed in the absence of symptoms. The expression of other genes such as acidic cellulase and methyltransferase2 was diminished in infected fruits.Huanglongbing, a highly destructive disease of citrus, threatens citrus-producing areas worldwide. Previous studies monitored transcriptional changes in leaves on a large scale using microarrays to investigate host responses and reveal the mechanisms underlying disease development. The present work focuses on transcriptional regulation in fruit peel to analyze the response to CaLas infection at different disease stages. Next-generation sequencing technology , as used here, can find differential expression of an increased number of transcripts, many of which may not be present in EST databases or represented in microarrays. NGS data can be used for specific transcriptome assemblies that become resource datasets for annotation of genomes and annotation of differentially regulated genes and proteins analyzed using any ‘‘omic’’ technique. However, 30 planter pot the absence of a completed genome sequence limited the advantages of RNA-Seq technologies. The aim of this work was to provide data using NGS technology for a comprehensive analysis of metabolic changes in fruit induced by HLB disease.

These findings will uncover the fruit disorder mechanisms and facilitate development of short-term therapeutic strategies for already-infected trees. Toward this end, the experimental design included four types of fruit: healthy control fruit from an HLB-free orchard, apparently healthy fruit from non-symptomatic trees in an orchard affected by HLB, and asymptomatic and symptomatic fruit from infected symptomatic trees in the affected orchard. This design made it possible to identify differentially expressed genes at different disease stages. A comparison between apparently healthy and asymptomatic fruit revealed genes induced early in disease development. Comparing asymptomatic and symptomatic fruit identified genes involved in the host response during disease progression. Comparing symptomatic and apparently healthy fruit revealed host response genes related to the presence of HLB symptoms. Healthy fruit from the HLB-free location were also compared to the three sample types from the infected location. Differences in gene expression in these comparisons are likely to result from environmental and agronomic variability due to the difference in location, in addition to the effects of HLB. A range of 1154 to 1762 differentially regulated genes were found using RNA-Seq comparing the three categories of fruits from the infected orchard with those taken from a location free of HLB . In qRTPCR analysis, 31 of 33 differentially regulated genes confirmed the pattern of expression found by RNA-Seq. Comparisons among the three fruit categories in the infected orchard resulted in fewer differentially regulated genes . This was expected, since samples within the same orchard grew under similar environmental and agronomic conditions. A similar contrast between samples from the infected and uninfected orchards is seen in principal component analysis. The transcripts that most strongly characterize the asymptomatic and apparently healthy samples are common between the two samples.

The symptomatic and uninfected orchard samples are both distinct groups and widely separated in the biplot . A visual summary outlines the most important transcriptional changes in the networks among genes, pathways, and cell functions in the fruit peel . Gene set enrichment analysis identified several pathways significantly affected by HLB as symptoms appear, considering both within-tree and between-tree comparisons, such as those for phenylpropanoids, starch and sucrose metabolism, carbon fixation, ascorbate, and alpha-linolenic acid . Other pathways were also affected by HLB in the two comparisons within the same orchard. Transcripts encoding different subunits of the photosystem II reaction center and cytochrome b6-f complex subunit were more abundant in symptomatic HLB-infected fruit than in healthy fruit. Photosynthesis is central to all aspects of plant biology, since it provides energy for growth and reproduction, but its regulation by biotic and abiotic stresses is still unclear. The induction of photosynthetic light reactions in the fruit is consistent with the observation that symptomatic HLB-infected fruit often remains green. The retention of green color and increase of photosynthesis reactions is probably linked to the lower amount of ethylene detected insymptomatic fruits. Photosynthesis is usually downregulated by pathogen attacks. The upregulation of photosynthetic reactions in the fruit does not contradict this common consideration because CaLas infections typically occur in young leaves. The transcriptomic changes observed in the fruit are probably linked to the source-sink disruption caused by leaf infections. Protein degradation and modification pathways were significantly changed by CaLas infection, as shown by the upregulation of genes such as C3HC4-type ring finger proteins involved in ubiquitin-mediated degradation. Interestingly, heat shock proteins HSP82 and HSP70, highly interactive proteins in the PPI network inferred in citrus, were down-regulated at different stages of the disease. Heat shock proteins are highly conserved proteins induced in cells subjected to elevated temperatures or other environmental stresses.

These proteins act as molecular chaperones to stabilize, reduce misfolding, or facilitate refolding of proteins that have been denatured during stress events. In plant cells, HSP70 and HSP90 are involved in signal transduction leading to plant defense responses. Both proteins interact with a salicylic acid-induced protein kinase and their silencing affected the hypersensitive response in Nicotiana benthamiana while inducing non-host resistance to Phytophthora infestans. HSP90 also modulates the innate immune responses involving gene-for-gene specific interactions, acting as a scaffold protein in a complex that mediates signal transduction. Based on these findings, we speculate that down-regulation of heat shock proteins observed at different stages of HLB disease might increase protein misfolding in the fruit peel. Genes encoding ATP synthase gamma and delta chains were also induced in symptomatic fruit, supporting the idea that CaLas may act as an energy parasite by scavenging ATP from its host with a pathogen-specific ATP/ADP translocase. Indeed, the recently sequenced genome of CaLas revealed the presence of an ATP/ADP translocase in addition to ATP synthase. ATP scavenging may be a possible mechanism of pathogenicity, affecting the fruit peduncle, columella, and seed coat. Photosynthesis regulation in infected fruit peel may also reduce transport of sugars and ions such as nitrate, sulfate, and potassium. In this study, several differentially expressed genes were associated with transport of ions, including ammonium, sulfate, and phosphate . Inorganic ions can modify sugar metabolism and photosynthesis. Polarized vesicle trafficking, transport, and secretion of plant materials are associated primarily with non-specific resistance during host-pathogen interactions. ABC transporters play important roles in this process, since they are also involved in virulence, host range, and symptom elicitation. Citrus proteins related to sugar and PDR/ABC transporters implicated in secretion of antimicrobial terpenoids were specifically induced by HLB at the symptomatic stage . Interestingly, among the genes identified in the CaLas genome were those for phosphate and zinc uptake into the cell. This implies that mineral uptake by the pathogen may be enhanced due to induction of endogenous genes as well as host genes. However, plastic growers pots phloem necrosis induced by CaLas contributes to the impaired nutritional transport functions and source-sink communication observed in this study. Starch accumulation in leaf chloroplasts of sweet orange trees infected with CaLas has previously been demonstrated and is characteristic for HLB. Genes encoding the large subunit of ADP-glucose pyrophosphorylase, the key enzyme catalyzing the first and limiting step in starch biosynthesis, were up-regulated in infected leaves. Interestingly, a gene encoding the large subunit of glucose-1-phosphate adenylyltransferase was downregulated in infected and symptomatic fruit in the present study. Also of interest are contrasting patterns of expression for different isoforms of starch synthase and starch cleavage genes, leaving the dynamics of starch accumulation in the fruit unclear . This is in contrast to experimental observation of starch accumulation in infected leaf tissues. In leaves, this accumulation was linked to up-regulation of genes involved in starch biosynthesis and down-regulation of its conversion to maltose. It has been hypothesized that starch accumulation is an effect of phloem plugging/necrosis during HLB infection, although it usually occurs before these symptoms are visible. Differences in starch accumulation are probably linked to the different types of organs: mature leaves are ‘‘source’’ and fruit are ‘‘sink’’. However, further analysis of fruit starch accumulation must be performed to validate this hypothesis.

An increased abundance of transcripts for genes involved in the first steps of glycolysis and sucrose metabolism was observed in fruits from infected trees. In the cytosol, an invertase gene was upregulated in asymptomatic and symptomatic fruit, affecting the sugar balance and communication between sink and source tissues. We speculate that this leads to increased glucose and fructose and decreased sucrose in fruit cells, which should be further validated with carbohydrate analysis. It is important to note that elevated glucose and fructose has been demonstrated in citrus leaves infected with CaLas. Interestingly, different types of invertases were up-regulated in leaves and fruits . Over-expression of yeast invertase in the cell wall of transgenic tobacco disrupted sucrose export, allowing soluble sugars and starch to accumulate, which consequently inhibited photosynthesis and resulted in stunted growth and bleached or necrotic leaf areas. As previously suggested for leaves, it is possible that the differential expression of key genes involved in sucrose and starch metabolism, as observed in CaLas-infected citrus fruit, might affect the osmotic potential and induce plasmolysis, thus altering the ripening process and producing typical HLB symptoms. However, further analysis of sugar concentrations will be necessary to clarify the causes and effects of disease symptoms in the fruit. Other studies on CaLas-infected leaves have shown increased sucrose and glucose, but not fructose. In fruit, it is possible that altered fructose and glucose concentrations might be responsible for physiological disorders and affect source-sink relationships with leaves. The gene set enrichment analysis confirmed that sucrose and starch metabolism were highly affected by the disease. Integrated analysis of leaf and fruit data indicates that sugar and starch metabolism play a key role in the metabolic dysfunction induced by HLB disease. However, few studies have been conducted to address the effects of altering sugar metabolism on resistance to pathogen infections. Invertase plays a key role in the activation of stress responses and may function as an extracellular indicator for pathogen infection. Indeed, transgenic plants overexpressing sugar metabolism enzymes such as a heterologous invertase from yeast have helped clarify source-sink relationships. The expression of different viral movement proteins in transgenic plants and the resulting effects on photosynthesis, carbohydrate accumulation, and partitioning emphasize the importance of sugars in activating defense responses against biotic attacks. Sink metabolism may be essential to satisfy the energy requirements of activating the cascade of defense responses. Interestingly, sucrose synthase was also more abundant in symptomatic fruit while sucrose transporter genes were downregulated by HLB. The Genevestigator database, indicated that these proteins may be down-regulated by hormones such as ethylene, methyl-jasmonates, and indol-3-acetic acid. The concept of sucrose metabolism regulators as a potential target for HLB therapeutics is intriguing. Increasing evidence indicates an extensive cross-talk between sugar, hormone, and light signal transduction networks in plants. Hormone pathways were significantly altered in fruit peel inresponse to CaLas infection. Two genes involved in auxin synthesis, GH3.1 and GH3.4, were induced in affected fruit. Gibberellin regulation has been observed in other fruit disorders such as albedo breakdown disorder and applications of GA3 before fruit color break can reduce the occurrence of some fruit disorders. It is possible that sugar metabolism changes observed in the fruit might be linked with the down-regulation of gibberellins that regulate energy and carbohydrate metabolism. Previous studies have demonstrated that regulation of gibberellic acid-induced gene expression is affected by sugar and hormone signaling. That cytokinins play a role in sugar regulation has been demonstrated. Therapeutic approaches using small-molecule hormones such as cytokinins and gibberellins may allow modification of fruit metabolism to mitigate the negative impact of HLB on fruit quality and productivity.Ethylene regulates a variety of developmental processes and stress responses in plants, including seed germination, cell elongation, senescence, fruit ripening, and defense. Nonetheless, ethylene can promote either disease resistance or susceptibility, depending on the host–pathogen interaction. In our study, considerable changes were observed in the transcriptional profiles of genes related to ethylene biosynthesis and signal transduction. ACC synthase and ACC oxidase play pivotal roles in ethylene biosynthesis and their expression is often affected by pathogen attack. It was unclear how ethylene concentration changes in fruit in response to HLB.

One of the reasons for this could be the nature of phenotypic complexity which is the result of reticulated interactions among many different physiological and cellular processes and environmental conditions . We used genetic and phenotypic analyses in the tomato IL’s, coupled with meta-analyses of existing data, to identify several co-regulatory relationships between carbon metabolism and leaves . This study proposed that leaf shape may affect the sugar content of tomato fruit through developmental and photosynthetic mechanisms. We used these relationships in directed experiments to explore additional avenues for fruit quality improvement.Previous studies have shown that there is a direct regulation of yield and BRIX in tomato through leaf shape by modeling these relationships using PLS-Path Modeling . This correlation was performed in heirloom cultivars which retain a large amount of genetic diversity compared to commercial varieties. This can make identifying the causative gene/s difficult. Another study utilizing monogenic mutants and their isogenic backgrounds showed a correlation between decreased vascular density and BRIX in tomato fruit. Here we used 17 tomato introgression lines , backcrossed IL , and sub IL’s to identify a specific gene which may influence Brix and yield through regulation of leaf shape and vascular density, bHLH032. bHLH032 is a SPATULA like transcription factor which when knocked out via CRISPR resulted in plants with decreased vascular density and increased BRIX x Yield .Leaf shape for these lines was measured and compared to their BRIX and yield traits from the same plants. Approximately 7637 leaflets were measured over two growing seasons, and their shape characterized using PCA analysis .

Nine lines showed significantly rounder leaflets compared to M82, including BIL 260, sub ILsub IL 4-3-4, BIL 063, blueberry in container and sub ILsub IL 5-4-1 . Additionally, two lines, sub ILsub IL 8-1-3 and IL 9-1-2, were significantly narrower than M82, with leaflets of the remaining lines similar in shape to M82 . BIL 260 and sub IL 4-3-4 had improved yield, and BIL 260 had improved BRIX at terminal harvest . Of the remaining lines, BIL 338 and BIL 378 were trending higher in yield than M82 at terminal harvest but were not significantly different . The inverse relationship between BRIX and yield has long been established , however BIL 260 breaks this relationship with an increase in both traits with yield greater than M82 by approximately 3-fold and increased BRIX . sub IL 4-3-4 also had an increased yield at just over 2 times that of M82 but had no significant difference in BRIX from M82 . To further quantify the total output of BIL 260 and sub ILs 4-3-4, they were grown with M82 over three independent field seasons, and the BRIX and yield of their terminal harvest multiplied to obtain the BY value index, a composite value which accounts for variations and extreme values in either measurement . The BY of BIL 260 is significantly higher than that of M82 at approximately 20, while M82 has a BY of 14 . The BY of sub ILs 4-3-4 was elevated but not significantly different from M82, most likely due to only an increase in yield and not BRIX .Because leaves are the primary site of photosynthesis, and responsible for most of the sugar production in plants, we checked to see if the photosynthetic rate , and stomatal conductance were altered in these lines. Several lines showed decreased photosynthesis compared to M82 , but only BIL 260 had increased photosynthesis . This increase is small but significant despite the large range of photosynthesis found in M82 . Several lines had reduced gst compared to M82, corresponding to lines which had decreased photosynthesis .

These same lines had reduced, but not significantly different, yields and BRIX values compared to M82 with the exception of BIL 338 and BIL 378 which each had slightly higher yield values . Both BIL 260 and sub IL 4-3-4 had similar gst as M82, despite the increase in photosynthesis in BIL 260 . Finally intrinsic water use efficiency was calculated for M82, BIL 260, and sub IL 4-3-4, with both introgression lines having significantly higher values suggesting both lines utilize water more efficiently than M82 . How this may impact the other measured physiological traits was not specifically studied in this research.To further understand the sugar usage in BIL 260 and sub IL 4-3-4 we analyzed sugar and starch mobilization and transport in leaves. This measurement was done at two-hour intervals across 24 hours for each of the three lines that were the focus of a detailed analysis, to identify time points where differences in their sugar and starch usage may occur . Figure 2d shows the sugar content of the leaves for all three lines measured as μmols per gram fresh weight. Much of the increase in sugar took place between 7am and 7pm hours, as would be expected as these were daylight hours , and for all three lines the concentration and change in sugar content was similar . However, between 1 am and 5 am there was an initial decrease in sugar content in M82 but then a subsequent large increase. In contrast, leaf sugar concentration in BIL 260 and sub IL 4-3-4 continued to decrease over this time period, suggesting that the sugar was either being utilized in the leaf or exported out of the leaves . Figure 2e shows the starch content from the same leaves and at the same time points. During daylight hours the change in starch content in the leaf is similar, though much higher in M82 at solar noon and interestingly all three genotypes have a decrease in starch content during the 1am to 5am time period .

This decrease in starch content and the concomitant increase in sugar content over this time in M82 suggests starch mobilization to sugar for usage in the leaves as no photosynthesis is taking place at this time . However, BIL 260 and sub IL 4-3-4 had a continued decline in sugar content of their leaves despite similar starch mobilization to that found in M82, which suggests that either the sugar is being utilized in the leaves at a more rapid rate or is being exported from the leaves at an increased rate . Despite the decrease in sugar content in BIL 260 and sub IL 4-3-4 being similar, BIL 260 has less sugar at 5am than either M82 or sub IL 4-3-4, likely due to even higher export rates .BIL 260 and sub IL 4-3-4 are both introgression lines which contain a small portion of Chromosome 4 from the S. pennelli genome in a majority M82 background. Because they both have yield difference but only BIL 260 has increased fruit BRIX and lower vascular density, we performed Whole Genome Sequencing to determine the exact location of the introgressions. In both lines the introgression is limited to chromosome 4, and there are no other introgressions present in the genome . The introgression spans the centromeric region and contains approximately 800 genes in each. However, there are a small number of genes introgressed from S. pennellii that are uniquely present in BIL 260 but not sub IL 4-3-4 on both the 5’ and 3’ ends of the introgression . On the 5’ end of the introgression there are 75 additional genes introgressed in BIL 260, while three additional genes are present on the 3’ end. Among the 78 additional genes from S. pennellii three are transcription factors, specifically bHLH032 , R2R3MYB20 , and a GRAS transcription factor . We hypothesized that these genes could be responsible for the differences seen between BIL 260 and sub IL 4-3-4 in factors such as yield, BRIX, vascular density, plastic planters bulk and sugar transport. As such we performed an RNA-Seq analysis for these lines and M82 covering three time points , and three tissue types in a field setting . The RNA-Seq data was split by tissue type and analyzed across all three time points. UMAP dimensionality reduction and Mean Shift clustering were performed on the DEGs for each tissue type . To determine the tissue type of most interest we looked for the three unique transcription factors from BIL 260 in the DEG lists and then for their position in the clustering. For SAM tissue the bHLH032 transcription factor was a DEG and present in clustering but the other transcription factors were not . In mature leaves bHLH032 was again a DEG present in the clustering but the other two were not . Young leaves, defined as those leaves fully developed but not expanded, had all three of these transcription factors of interest present in the DEG list .

The GRAS transcription factor was within Cluster 6 which had GO enrichment for protein folding and intracellular protein transport. The R2R3Myb was found within Cluster 31 which had no significant GO enrichment . bHLH032 was found within Cluster 19 which had GO enrichment terms relating to sugar metabolism and extracelluar regions . Additionally, the ortholog of bHLH032 in Arabidopsis is Abnormal Shoot 5 , which when over expressed causes upward curled leaves and increased vascular density . It is also shown thatABS5 interacts with Lonesome Highway to initiate vascular development in combination with TMO5 . Given the leaf and vascular changes observed in BIL 260 along with the increased BRIX and yield, we chose to pursue cluster 19 and the bHLH032 TF. Gene co-expression analysis was done on cluster 19 to identify any differences between BIL 260 and M82 . The M82 network for cluster 19 contains bHLH032 as a peripheral gene connecting to the network through Solyc08g067030 . A BLAST search of the protein sequence for this gene showed three potential Arabidopsis orthologs, At4g32460 , At5g11420, and At5g25460 . All three genes have expression in either mature or developing leaves, and BDX is involved in vascular development . Additionally, a phospolipase-A2 like gene was also correlated with the BDX/DGR2 gene . An aldose-1-epimerase , is also present and could indicate a connection with sugar metabolism or processing. The BIL 260 network did not contain the bHLH032 gene but did still have BDX/DGR2 connected to the central network . The aldose-1-epimerase was no longer within the central part of the network, but a peripheral gene, and not connected BDX/DGR2 . The same was found for the phospholiapse-A2 like gene, indicating a large rearrangement of the network including the loss of bHLH032 . The absence of bHLH032 from the BIL 260 network suggested either a loss of function or reduction in expression for the S. pennelli version of this gene, and so we analyzed the expression values found in the RNA-Seq data . The overall expression of bHLH032 in BIL 260 was lower than that in M82, with a p-value of 0.06 across all weeks. At week 10, when plants were flowering and beginning early fruiting, the expression of bHLH032 in young leaves was much lower than that found in M82 . This was confirmed by the eFP browser for tomato which also showed that in S. pennellii bHLH032 has a much lower expression than M82 .There are approximately 800 genes found within the BIL 260 introgression from S. pennellii, and as such the differences in BY, leaf shape, and vasculature could be a result of other genes within the introgression and not the bHLH032 despite the gene co-expression network results. To determine if this was the case we back-crossed BIL 260 with M82 to break up the introgression region. The F1 populations were grown in the greenhouse for seed, and then 400 F2 plants were taken to the field for characterization. The BY for all lines, including the parent lines M82 and BIL 260, were measured . There was a wide range of BY’s with the majority of plants falling within the parent plants range, but transgressive phenotypes were seen as both lower and higher than either M82 or BIL 260 . The vegetative biomass was also measured and compared to fruit mass to see if there was an equivalent change in both, but vegetative biomass remained consistent across all F2 plants while yield was highly variable . From these data we selected lines that had both high and low BY as well as round and narrow leaflets to grow the F3 and F4 populations . Additionally, all 400 lines were sequenced using GT-Seq .

Six seedlings of each genotype were planted per pot for each replicate. The 76 IL’s were divided into four cohorts of 20 randomly assigned genotypes. These cohorts were placed across four temporal replicates in a Latinsquare design as described in . The seedlings were harvested 5 d after transplanting . Cotyledons and mature leaves .1 cm in total length were excluded, and remaining tissues above the midpoint of the hypocotyl were pooled, for all individuals in a pot, into 2-mL microcentrifuge tubes and immediately frozen in liquid nitrogen. Two IL’s, IL7.4 and IL12.4.1, were not included in the final analysis due to seed contaminations.Seeds 76 IL’s along with the parents were sterilized using 70% ethanol, followed by 50% bleach, and finally rinsed with sterile water. This experiment was replicated three times each in 2011 and 2012. Ten to 12 seeds of each IL were sown into Phytatray II containers with 0.53 Murashige and Skoog minimal salt agar. Trays of each IL were randomly assigned to either a sun or shade treatment consisting of 110 mmol PAR with a red to far-red ratio of either 1.5 or 0.5 at 22°C with 16-h-light/8-hdark cycles for 10 d. Three genotypes were excluded from the analyses due to poor germination or their necrotic dwarf phenotypes . After 10 d, seedlings were removed from the agar and placed onto transparency sheets containing a moistened kimwipe to prevent dehydration and scanned using an Epson V700 at 8-bit grayscale at 600 dpi. Image analysis was carried out using the software ImageJ . For hypocotyl length analysis of backcross inbred lines between S. pennellii and S. lycopersicum cv M82, growing berries in containers seeds were sterilized in 50% bleach and then rinsed with sterile water.

The seeds were then placed in Phytatrays in total dark at room temperature for 72 h and then moved to 16 h light/8 h dark for 4 d. Seedlings were transferred to soil using a randomized design and assigned to eithera sun or shade treatment for 7 d. Images were taken with an HTC One M8 Dual 4MP camera and hypocotyl lengths measured in ImageJ using the Simple Neurite Tracer plugin.RNA-seq libraries were prepared and the reads were preprocessed as de- scribed in Chitwood et al. and are outlined here. mRNA isolation and RNA-seq library preparation were performed from 80 samples at a time using a high-throughput RNA-seq protocol . The prepared libraries were sequenced in pools of 12 for replicates 1 and 2 and in pools of 80 for replicates 3 and 4 at the UC Davis Genome Centre Ex- pression Analysis Core using the HiSeq 2000 platform . Preprocessing of reads involved removal of lowquality reads , trimming of low-quality bases from the 39 ends of the reads, and removal of adapter con- tamination using custom Perl scripts. The quality-filtered reads were sorted into individual libraries based on barcodes, and then barcodes were trimmed using custom Perl script.Mapping and normalization were done on the iPLANT Atmosphere cloud server . S. lycopersicum reads were mapped to 34,727 tomato cDNA sequences predicted from the gene models from the ITAG2.4 genome build . A pseudo reference list was constructed for S. pennellii using the homologous regions between S. pennellii scaffolds v.1.9 and S. lycopersicum cDNA references above. Using the defined boundaries of IL’s, custom R scripts were used to prepare IL-specific references that had the S. pennellii sequences in theintrogressed region and S. lycopersicum sequences outside the introgressed region. The reads were mapped using BWA using default parameters except for the following that were changed: bwa aln: -k 1 -l 25 -e 15 -i 10 and bwa samse: -n 0.

The bam alignment files were used as inputs for express software to account for reads mapped to multiple locations . The estimated read counts obtained for each gene for each sample from express were treated as raw counts for DE analysis. The counts were then filtered in R using the Bioconductor package EdgeR version 2.6.10 such that only genes that had more than two reads per million in at least three of the samples were kept. Normalization of read counts was performed using the trimmed mean of M-values method , and normalized read counts were used to identify genes that are differentially expressed at the transcript level in each IL compared to cv M82 parent as well as between two parents, S. pennellii and M82. The DE genes for each IL were compared to those between the two parents to identify genes that were differentially expressed for the IL but not for S. pennellii compared to cv M82. Those genes were considered to show transgressive expression pattern at the transcript level for the specific IL, whereas other DE genes were considered to show the transcript expression similar to S. pennellii.RNA-seq libraries were prepared and the reads were preprocessed as de- scribed in Chitwood et al. and are outlined here. mRNA isolation and RNA-seq library preparation were performed from 80 samples at a time using a high-throughput RNA-seq protocol . The prepared libraries were sequenced in pools of 12 for replicates 1 and 2 and in pools of 80 for replicates 3 and 4 at the UC Davis Genome Centre Ex- pression Analysis Core using the HiSeq 2000 platform . Preprocessing of reads involved removal of low-quality reads , trimming of low-quality bases from the 39 ends of the reads, and removal of adapter contamination using custom Perl scripts. The quality-filtered reads were sorted into individual libraries based on barcodes, and then barcodes were trimmed using custom Perl script.Mapping and normalization were done on the iPLANT Atmosphere cloud server . S. lycopersicum reads were mapped to 34,727 tomato cDNA sequences predicted from the gene models from the ITAG2.4 genome build .

A pseudo reference list was constructed for S. pennellii using the homologous regions between S. pennellii scaffolds v.1.9 and S. lycopersicum cDNA references above. Using the defined boundaries of IL’s, custom R scripts were used to prepare IL-specific references that had the S. pennellii sequences in the introgressed region and S. lycopersicum sequences outside the introgressed region. The reads were mapped using BWA using default parameters except for the following that were changed: bwa aln: -k 1 -l 25 -e 15 -i 10 and bwa samse: -n 0. The bam alignment files were used as inputs for express software to account for reads mapped to multiple locations . The estimated read counts obtained for each gene for each sample from express were treated as raw counts for DE analysis. The counts were then filtered in R using the Bioconductor package EdgeR version 2.6.10 such that only genes that had more than two reads per million in at least three of the samples were kept. Normalization of read counts was performed using the trimmed mean of M-values method , and normalized read counts were used to identify genes that are differentially expressed at the transcript level in each IL compared to cv M82 parent as well as between two parents, S. pennellii and M82. The DE genes for each IL were compared to those between the two parents to identify genes that were differentially expressed for the IL but not for S. pennellii compared to cv M82. Those genes were considered to show transgressive expression pattern at the transcript level for the specific IL, whereas other DE genes were considered to show the transcript expression similar to S. pennellii.Transcript level patterns were correlated with three phenotypes collected from the IL’s along with the parents. Normalized estimated read counts with 3 to 4 independent replicates per IL were log2 transformed prior to the analyses. Leaf number and complexity were collected from the IL’s as outlined in Chitwood et al. under both sun and shade treatments. Hypocotyl lengths were measured as detailed above. To test whether the transcript level for a given gene was correlated with a particular phenotype, blueberry containers boostrapping analyses were performed. Transcript levels and phenotype data were randomly per- muted using the sample function against IL and then merged. For each analysis, 1,000 replications were performed and the P values were calculated from the Spearman’s rho value distributions. P values were adjusted for multiple comparisons using the BH correction . Significant correlations were identified as those with an adjusted P value, 0.05, and the mean rho value was used to designate the correlation as either positive or negative . All analyses were implemented using the statistical software R and custom scripts .eQTL mapping analyses were performed to determine whether the transcript level of a gene is correlated with the presence of a specific introgression from S. pennellii into S. lycopersicum cv M82. This correlation was examined at the level of “bin,” with a bin defined as a unique overlapping region between introgressions. Examining eQTL at the bin level enables those eQTL to be mapped to considerably smaller intervals than the IL’s themselves . eQTL mapping analyses were performed on the normalized estimated read counts with 3 to 4 independent replicates per IL, which were log2 transformed prior to the analyses. To test whether the transcript level for a given gene is correlated with the presence of a particular bin, a Spearman’s rank correlation test was used with ties resolved using the midrank method. P values were adjusted for multiple comparisons using the BH correction . Significant eQTL were identified as those with an adjusted P value, 0.05, and Spearman’s rho was used to designate the eQTL as up or down .

Significant eQTL were also designated as cis if the gene was located on the bin with which it is correlated; trans if the gene was correlated with a bin that is neither the bin it is on nor a bin that shares an overlapping IL with the correlated bin; or chromo0 if the gene lies in the unassembled part of the genome. When a gene has a designation cis- eQTL, and a secondary correlation was found with a bin that shares an overlapping introgression, this secondary correlation was not designated as an eQTL. When a gene does not have a designated cis-eQTL and a correlation was found with a bin that shares an overlapping introgression, this correlation was designated as a trans-eQTL. All analyses were implemented using the statistical software R and custom scripts .t-SNE or t-distributed stochastic neighbor embedding is a nonlinear dimensionality reduction method, which faithfully maps objects in high dimensional space into low dimensional space . Crowding is avoided through the longtailed t-distribution, which forces nonneighbor clusters farther away from each other in V-space than those clusters actually are in H-space . The ex- aggerated separation of non-neighboring clusters improves 2D resolution, allowing identification of novel groupings not readily apparent in other clustering methods. However, this method is resource intensive, and with higher dimensionality, the number of genes that can be analyzed is limited. We have used Barnes-Hut-SNE, a newer implementation of t-SNE that greatly increases the speed and number of genes that can be analyzed, for the present analysis . BarnesHut-SNE accomplishes this efficiency through the use of a Vantage Point tree and a variant of the Barnes-Hut algorithm . For clustering, 2D maps were generated using a perplexity of 30 and without the initial PCA step from the Barnes-Hut-SNE R implementation . Theta was set to 0.3 based on van der Maaten to maintain an accurate dimensionality reduction without sacrificing processing speed.The DBs can algorithm was used to select modules from the Barnes-Hut-SNE results . This algorithm had the advantage of both selecting modules and removing any genes that fell between modules. The scanning range and minimum seed points were selected manually and used to deter- mine if any one point is a member of a cluster based on physical positioning within the mapping relative to neighboring points. A minpts of 25 was used to capture smaller modules on the periphery, and an epsilon of 2.25 was used to avoid the overlapping of internal and closely spaced modules.Box plots were generated from normalized transcript abundance values for each module. The ribbon plot was generated from correlated abundance values from leaf development and photosynthesis related modules. These plots were generated using ggplot form the ggplot2 R Package . The median transcript levels of the genes mapped to a module were calculated for each IL and replicated for all modules.

Specifically, a principal coordinate analysis of weighted UniFrac distances indicated significant clustering of individual microbial communities by phenological stage . In a pairwise comparison of community compositional differences between each phenological stage, the leaf bacteriome had the greatest number of significant adjusted P values, with 21 of the 21 pairwise comparisons being significantly different, followed by the leaf mycobiome . Rainfall, fertilizer applications, temperature, and irrigation hours fluctuated across our sampling period . Rainfall was sparse in this sample location , ranging from 0.00 to 2.55 inches each month , and total rainfall was a minor determinant of community structure across all four communities, explaining only 0.8 to 2.0% of the variation . Similarly, fertilizer application describes a small percentage of the variation in the data for all four communities examined. We evaluated temperature based on the average temperature and interactive effects it might have with water availability in order to capture the full range of conditions that could affect microbial community composition. Temperature had a minor impact on communities, as this factor describes only 0.6 to 2.8% of the variation in the data that include temperature as an interaction factor. In addition to phenology, interactions between phenology and sample year were a driving factor of leaf bacterial community composition, explaining 8.2% of the changes across the data . Taken together, these beta diversity analyses indicate that plant phenological stage was the major driving factor in community composition for bacterial and fungal communities associated with leaves and roots.

Significant compositional shifts were also visible at the phylum level, container growing raspberries particularly in the leaf bacterial community . Other covariates tested were minor or insignificant contributors to citrus-associated leaf and root microbiome composition.We identified core microbial taxa for each of our seven phenological stages. Our core bacterial and fungal leaf and fungal root microbiomes include genera that were greater than 0.01%, and core root bacteriome included genera greater than 0.1%, relative abundance in at least 75% of the samples within a phenological stage. All of our downstream analyses used genera that met our core taxa cutoffs in at least one phenophase. We assessed our core taxa and separated them into three categories: high stability, defined as core member of six or more phenophases, medium stability, core member of three, four, or five phenophases, or low stability, core member of two or fewer phenophases. We determined that of the identified core there were 3 leaf bacterial, 8 leaf fungal, 62 root bacterial, and 22 root fungal core genera that had high stability across phenophases . This suggests that both bacterial and fungal root communities have a substantially greater number of consistent or stable microbial features across the developmental cycle. However, our experimental design did not differentiate between endophytes versus epiphytes and, thus, may have missed some fine resolution microbial community shifts occurring between the endosphere and episphere. There were two bacterial and one fungal genera that were highly stable in both roots and leaves . A phylogenetic analysis of the core genera indicates that both bacterial and fungal root communities were rich in highly stable and phylogenetically diverse core taxa . Root core genera from the bacterial clade Alphaproteobacteria and the fungal family Pleosporomycetidae were all or nearly all binned as highly stable, indicating that genera in these clades were consistently high in relative abundance across all phenological stages.

Medium- and low-stability core genera appear randomly dispersed across the root community phylogeny, with no obvious patterns.However, leaf bacterial and fungal core community phylogenetic trees contained high, medium, and low stability patterns at the class and phylum levels . All core genera in the fungal class Tremellomycetes had medium to high stability. In contrast, all core genera in the fungal class Sordariomycetes had low stability across phenophases and met the defined core cutoffs only during fruit set or mature fruit stages. The leaf taxa within the bacterial class Gammaproteobacteria consisted of genera with high, medium, and low stability across the phenophases. Interestingly, all theGammaproteobacteria were core members of the full flowering or floral bud development microbiomes regardless of their stability in other phenophases. Another distinct phylogenetic pattern observed in the leaf community was genera in the bacterial phylum Actinobacteria that had low or medium stability across all phenophases. However, 95.0% of core genera in the Actinobacteria clade were core during fruit set and/or fruit development. The only exception to this within the Actinobacteria clade was Bifidobacterium, which was associated only with full flowering and was not a core member of fruit set or fruit development microbiomes . Lastly, the leaf bacterial class Betaproteobacteria contains low- to medium-stability core genera with the most dispersed stage associations. Overall, these data indicate that root bacterial and fungal communities have greater stability across phenophases than those of leaves . Additionally, core taxa had phylogenetically related trends within the high-, medium-, and low-stability classifications, indicating that conserved, vertically descended microbial traits may play a role in determining bacterial and fungal associations across phenophases, particularly in above ground leaf tissue.We completed a genus-level differential relative abundance analysis on our list of core taxa that were $0.01% relative abundance and $75% prevalence in one or more phenophases.

Our differential relative abundance analysis can determine finer-scale phenophase associations beyond just classification as a core microbiome member by looking for increases in relative abundance, proportionate to other members of the microbial community , during specific phenophases. Ecologically dominant taxa are predicted to have a proportionately larger contribution to community function. Among all the phenophases, those associated with flowering had striking microbial enrichments, particularly among the leaf bacteria. Acinetobacter was a core member of five phenophases but was significantly enriched during full flowering compared to other phenophases . Acinetobacter had a gradual enrichment from flush and floral bud development to full flowering. This gradual enrichment signature indicates that Acinetobacter was present throughout the year but has a high temporal turnover rate that is in sync with the transitions from flush to floral bud development and then to full flowering. We also observed bacteria that were sharply enriched during full flowering rather than undergoing gradual enrichments over the phenophases that lead up to full flowering . These include Snodgrassella, Frischella, Gilliamella, and Bifidobacterium . The sharp enrichment patterns during full flowering suggest that these taxa were introduced into the community via a dispersal event.We also identified bacterial leaf genera that had significant depletions during floral bud development and/or full flowering . Four Actinobacter genera, Corynebacterium, Dietzia, Georgenia, and Ornithinimicrobium, were significantly depleted during floral bud development and full flowering . Bacillus, Methylobacterium, Romboutsia, and Sphingomonas also significantly decreased in relative abundance during floral bud development and/or full flowering . For all differentially abundant genera, including bacteria and fungi, across all phenophases, see Fig. S5 and Table S4.We performed a network analysis on the foliar bacterial communities from all samples. We focused on significantly enriched and/or depleted populations and populations with direct connections or putative first-degree interactions . The goal of this approach was to give a broad overview of bacterial interactions across phenophases and identify taxa that potentially interact with specific phenophase-enriched taxa and potentially play a role in observed seasonal community compositional shifts. Rhizobium, Sphingomonas, an unknown bacterium, an unknown Bacillaceae , Acinetobacter,and Romboutsia have the highest normalized betweenness centrality scores ranging from 0.110 to 0.187. Betweenness centrality is a proxy for influence within a network because it measures how often a particular node is the shortest connection or bridge between two other nodes. These high betweenness centrality scores and placement within the network indicate that these genera are potentially keystone taxa that may perform a stabilizing role in the microbial communities across phenological transitions and events . Groups of taxa connected by putative positive interactions cluster together to form distinct modules. These modules are separated by putative negative interactions. Our analysis organized bacterial taxa that were enriched in fruit set and fruit development into a single highly connective community module .

This suggests that fruit set- and fruit development-associated microbiomes are compositionally similar and few microbe-microbe interactions change during the transition from fruit set to fruit development. Leaf bacteria associated with flowering also formed a module within the network . Specific bacteria within the fruit set/development and flowering modules also interact with taxa that were enriched in the other four phenophases, blueberries in pots which cluster together into a third module . Overall, these predicted positive interactions represent inter- or codependent microbe-microbe relationships, and the putative negative interactions indicate potential direct or indirect competition. These predicted microbe-microbe interactions within the microbiome likely affect community compositionin addition to the exogenous influences of abiotic environmental conditions and biotic host physiological factors .The majority of studies examining how plant developmental stage affects the plant’s microbiome have focused on bacteria associated with the rhizosphere of annuals or herbaceous perennials such as maize , rice , sorghum , wheat , Arabidopsis , and Boechera . These important studies indicate that rhizosphere-associated microbiomes can shift in association with plant developmental stages in both domesticated and wild plants that have short-lived aboveground tissues. Studies of the endophytic xylem sap microbiome in grapevine, a deciduous perennial, also showed that microbial shifts were linked to changes in phenological stage . However, much less is known about how overall plant phenology affects above- and belowground microbiomes of evergreen woody perennials that have lifespans that can be decades long and can retain their leaves for multiple years, compared to annuals or deciduous perennials that produce and shed all their leaves each season. Here, we investigated microbiome compositional dynamics in above and below ground tissues of mature 20-year-old Citrus sinensis trees to determine if temporal microbiome fluctuations were associated with host phenological events. The unique contribution of our research was the separation of leaf development from tree phenology. We did this by analyzing the changes in the foliar microbiome on fully mature leaves, which developed in the leaf cohort from the previous year, in relation to the phenological stages of the current year. Thus, the leaves were exposed to the same starting inoculum, minimizing the bias of any potential priority effects . Our results indicate that the phyllosphere microbiome has an active and dynamic relationship with host phenology. More specifically, microbial shifts occurred as trees transitioned from the spring leaf flushing stage and entered flowering. The transition from spring flush to floral bud development and full flowering aligns with important transitions in source-to-sink transport of photosynthate in the tree . During foliar flushing periods, young leaves are a primary carbohydrate sink as they rapidly expand and mature. This source-to-sink transport of photosynthate shifts during floral bud break and development, when mature leaves transition to serve as source tissues to developing floral tissues that are also primary sink tissues. In addition to changes in source-to-sink transport, there are also significant changes in water dynamics within the canopy of the tree associated with full flowering. Flowers have the highest transpiration rate of the tree even compared to the leaves, which drastically increases the amount of water being transported into the overall canopy of the tree . Interestingly, the significant shift in overall foliar community composition from flushing to full flowering was not coupled with a change in species richness, indicating that the same taxa were present, just in different relative abundances in relation to one another. This demonstrates that foliar microbiome assemblage is changing in sync with tree physiology and development. Empirical data, including presence/absence and relative abundance, can also be used to infer patterns or microbial enrichments and/or depletions relative to othertaxa in the community, as well as ecological mechanisms that contribute to plant microbiome assembly, such as microbial species turnover and dispersal . Interestingly, microbial enrichment and depletion patterns of specific taxa suggest that microbial species turnover and dispersal events within the citrus microbiome occur in sync with phenological stage transitions. These enrichment/depletion patterns for specific taxa were more apparent in leaves than in the root compartment. Specifically, the bacterial genus Acinetobacter was enriched in leaves as trees transitioned from spring flush to floral bud development and peaked in relative abundance during full flowering, which is when mature leaves shift toward becoming source tissues for developing flowers and fruit. This may create a microenvironment that selects for an increase in relative abundance of these taxa when carbohydrate is translocating out of the leaves.

Several studies have compared hydrolyzable tannin profiles in developing pomegranate fruits. However, the fruit developmental stages were defined by different standards, such as days after fruit set/full bloom, physico-chemical properties, or physiological attributes of the fruit . Developing fruits of two cultivars, “Wonderful” and “Rosh Hapered,” grown in Israel were collected during a span of 8 or 10 weeks . Three major hydrolyzable tannins, gallagic acid, punicalin isomers , and punicalagins, were quantified in water extracts of the developing fruits. Fruits of “Ruby” grown in South Africa were harvested at five stages according to days after full bloom . Total hydrolyzable tannins in aril juice declined during the progression of fruit maturation, and were accompanied by decreases in ellagic acid and gallic acid . Relative amounts of hydrolyzable tannins in fruit peel, aril juice, and seed of developing pomegranate fruits were also investigated. Fruits of the Chinese cultivar “Taishanhong” were harvested at 10-day intervals for nine collections. Unicalagins, ellagic acid, and gallic acid were higher in fruit peel than aril juice and in seed; all three metabolites showed decreased accumulation in the three tissues during fruit development . When quantified by absorption of the methanolic extracts at 550 nm, total hydrolyzable tannins gradually decreased in fruit peels at low, low-medium, medium, plant pots with drainage and medium-high stages of the Spanish cultivar “Mollar de Elche.” In contrast, they were not detectable in aril juice at all stages.

In seeds total hydrolizable tannins increased at medium and then decreased at medium-high stages . Overall, despite the differences in the genetic background, growth conditions, harvesting scheme, and extraction and quantification methods, there is a consistent trend of decreasing hydrolyzable tannin accumulation in fruit peels, aril juice, and seed through pomegranate fruit development.To understand the impact of growth environment on hydrolyzable tannin profiles, fruit peel and aril juice hydrolyzable tannins were compared for 11 accessions grown in the Mediterranean or desert climate in Israel . Mediterranean climate promoted high levels of hydrolyzable tannins in aril juice in most of the accessions evaluated; in contrast, desert climate had a positive impact on hydrolyzable tannins in fruit peels . It was reported that the sweet/sour phenotype and environment interactions had the most influence on total tannin variations in aril juice of 10 commercial cultivars grown in four different regions in China, followed by the growth environment . There were negative correlations of overall average temperature with total polyphenol, total tannin, and punicalagin concentrations. The sweet/sour phenotype only accounted for 0.06% of the variations in tannins among different cultivars . The quality of aril juice under deficit irrigation was investigated in Spain . Three water regimes were applied to pomegranate trees at 75% evapotranspiration , 43% ETo , and 12% ETo . Water stress drastically decreased punicalagins, causing 30 and 70% reduction in moderate and sever stresses, respectively, in aril juices of fruits harvested from the corresponding trees . This study provided valuable information on the implications of water stress on the hydrolyzable tannin metabolism and the nutritional value of aril juice.Plants have developed diverse mechanisms to regulate their biological and metabolic processes via transcription factor regulatory networks .

Among the TF families, the basic leucine zipper family is present in all eukaryotes and is one of the largest and most diverse TF groups in higher plants. There are about four times more bZIP genes in the Arabidopsis genome than in the genomes of other model organisms such as Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster . Large numbers of bZIP TF family members have been found in many plant species including rice , maize , tomato , common wheat , sorghum , soybean , banana , cassava , grape , peach , strawberry , apple , rapeseed , radish , cucumber , tea plant , sweet potato, watermelon/melon , Chinese jujube , pepper , Chinese pear , poplar , quinoa and plum . The bZIP family is phylogenetically categorized into different groups, with different species having various members of homologs. For example, the Arabidopsis AtbZIP family members were systematically classified into 10 groups based on conserved motifs . Subsequently, a more complete classification was expanded into 13 groups, designated as A-J, M, and S . The tomato SlbZIPs were classified as nine clades . The cucumber CsbZIPs and sorghum SbbZIPs were separately categorized into six and seven groups . The bZIP family in both rice and maize has 11 groups which are the same as castor bean . The plum PmbZIP proteins were divided into 12 groups . Chinese pear PbbZIPs were categorized into 13 groups . Several interspecies clustering studies indicate that the S group found in Arabidopsis has especially high homology across different species , although some clades might be specific to Arabidopsis compared to peach, strawberry, and apple . These classifications, phylogeny, and homology analyses define the possible biological roles of bZIPs in green plant evolution .

Basic leucine zipper TFs orchestrate a diverse array of functions in multiple biological processes including flower development and pollen development , seed maturation , senescence , light signaling , anthocyanin and chlorophyll biosynthesis , nutrient signaling , hormone signaling such as salicylic acid, ABA, ethylene, auxin, and cytokinin , sugar signaling , and abiotic/biotic stress signaling in plants. Group S is the largest bZIP subgroup in several species such as Arabidopsis and safflower and comprises three to four even smaller subgroups. In this review, we focus on the well-studied S1-bZIP subgroup, whose members contain unique conserved upstream open reading frames in the 50 region of their transcripts and play important regulatory roles in many metabolic processes relating to fruit quality and stress responses. Our review aims to provide perspectives for further surveying the biological function, exploring regulatory mechanisms, and genome engineering the S1-bZIPs to obtain desirable traits for quality improvement in horticultural plants.Of the AtbZIPs, the 17 members of the S group are further separated into three subgroups based on homology: S1, S2, and S3 . The S1 subgroup in Arabidopsis contains five members: AtbZIP1, −2, −11, −44, and −53. Recent studies indicate that other species, including many horticultural plants, also have multiple members of the S1- bZIP subgroup . Like other bZIP members, those in the S1 subgroup are characterized by a conserved bZIP domain, composed of two functionally distinct motifs located on a contiguous α-helix. The basic region of −18 amino acids contains, sequentially, a nuclear localization signal and an invariant N-x7-R/K-x9 motif for DNA binding. This motif preferentially binds to the A-box, C-box, and G-box of target promoters which contain DNA sequences with an ACGT core . The leucine zipper comprises a heptad repeat of leucines or other numerous hydrophobic amino acids . Compared to other groups, members of the S group include the extraordinarily high number of eight hydrophobic amino acid repeats . The two subunits form a zipper structure that binds DNA to form dimers through interactions with the hydrophobic sides of the helices . Of three S subgroups, only members of the S1 subgroup show specific heterodimerization with C group bZIP proteins , whereas weak homodimerization within members of the S1 subgroup is detected . Phylogenetic analysis between S1 and C group bZIPs from angiosperms, gymnosperms, mosses, and algae suggests that the S1 and C groups evolved from a proto-S/C bZIP in algae species that homodimerized, which has since diverged into heterodimerizing pairs prior to the evolution of seeds plants .Besides their common structural features, S1-bZIPs are unique in that they have an unusually long 50 -leader sequence in the upstream region of the main open reading frame of the mRNA. This leader sequence contains several upstream open reading frames that encode small peptides . Among those, the second uORF is conserved and encodes a Sucrose Control peptide of 28 residues, plastic plants pots which regulates the translation of the mORF and reduces protein expression through a mechanism known as Sucrose-Induced Repression of Translation , which contributes to sucrosehomeostasis in the cells . Here, we summarize uORFs of the S1-bZIP subgroup from different horticultural plants, including banana , grape , apple , peach , cucumber , strawberry , petunia, and white pear . The regulation of gene expression involves different layers, including transcriptional and translational controls . Compared with transcriptional regulation, translational control allows more immediate responses to adjust protein expression and reprogram metabolism upon cellular signals or environmental stimuli . The translation process of mRNA includes four major steps: initiation, elongation, termination, and ribosome re-initiation . Translation initiation is the major step that determines the rate of protein biosynthesis and is regulated by multiple mechanisms . uORFs have been suggested to play a critical role in regulating the translation of the mORF . uORFs of S1-bZIPs are involved in the translational regulation in a SIRT manner .

The SC-peptide encoded by the uORF in the 50 leader region of AtbZIP11 is capable of repressing translation of the subsequent mORF in the presence of sucrose . High sucrose levels enhance ribosome stalling on the uORF, which results in poor translation of the mORF . The members of the Arabidopsis S1-bZIP subfamily show similar responses to sucrose. Translation of AtbZIP1, AtbZIP2, AtbZIP11, AtbZIP44 and AtbZIP53 is down regulated by sucrose . Transgenic seedlings with 35S:bZIP11 50 leader::LUC show significantly reduced luciferase activities when treated with sucrose while those incubated in media lacking sucrose show two- to three-fold higher luciferase activities . SIRT-mediating S1-bZIP orthologs exist in all seed plants .Previous research showed that amino acids such as serine, leucine, and tyrosine in the conserved peptide of uORF are essential for SIRT . However, it has been shown that expressing the gymnosperm 50 uORF sequence, which only contains the conserved leucine and tyrosine in Arabidopsis cells efficiently mediates the translational repression of the LUC reporter gene in response to sucrose . This study suggests that the SIRT mechanism most likely depends on structural conformation, but not on recognition of specific sequence motifs . Recently, interesting research conducted using gene-editing technology in strawberry demonstrated that uORFs are involved in regulating protein translation efficiency and sucrose content . In the study, to manipulate the SC-uORF of FvebZIPs1.1, the start codons of the uORF and the codons encoding a conserved pair of amino acid arginine within the SCpeptide were edited using the CRISPR/Cas9 system. Mutations in the start codons and the conserved C-terminal region of the SC-peptide significantly reduced translation of the SC-uORF. This consequently enhanced the translation efficiency of the downstream mORF. Seven novel alleles with C-to-T substitutions and small deletions within the uORF were identified. To test if phenotypic effects were additive in heterozygous and biallelic plants, 4000 T1 seedlings were generated by crossing the biallelic and homozygous T0 mutants to each other and to wild type. 35 novel genotypes were obtained in T1 and inherited in T2 generation. In comparison with wild-type fruits, the mutants had significantly higher levels of fructose, glucose, and total sugar contents, demonstrating that engineering the conserved SCuORF of FvebZIPs1.1 can increase the sugar content in strawberry . In addition, the citric acid content was slightly lower in the homozygous mutants than that in wild type. A continuum of gradual increase of sugar contents was generated in T1 by combining heterozygous, homozygous, and biallelic mutants, and inherited in T2 generation by propagating stolons of these T1 mutants, therefore confirming the transmissibility of novel genotypes and phenotypes from T1 to T2 by asexual propagation . Given that sugars can modulate multiple growth and development processes, the agricultural traits including leaf shapes, leaf areas, plant height, growth rates, pollination, fruit size and fruit weight were further evaluated in FvebZIPs1.1 uORF mutants. Remarkably, editing SC-uORF does not severely impair plant growth. The agricultural traits in FvebZIPs1.1 uORF mutants were similar to wild-type , whereas impaired phenotypes and retarded growth are observed in transgenic lines with the overexpression of AtbZIP11, tbz17, and FvbZIP11 mORF . Taken together, this suggests a broad application of editing uORFs of S1-bZIPs for quality improvement in horticultural plants.Amino acids are not only involved in plant response to stress but also influence fruit flavor . For example, asparagine is present in almost all fruits and determines fruit flavor and quality in a concentration dependent manner . Glutamate is responsible for “umami” or savory taste . Glycine, alanine, serine, threonine, proline, glutamine, and lysine are highly correlated with sweetness , while phenylalanine and tyrosine are bitter .

However, as discussed above, BER and NCED are both induced by drought. Therefore, a new study was necessary to trigger NCED gene expression independent on inducing BER directly, such as during drought conditions. Based on those difficulties, different promoters could be used to achieve the best results. The ethanol-inducible system is derived from the fungus Aspergillus nidulans in which the alcR gene encodes transcription factor ALCR that controls the activation of several structural genes, such as alcA . In the absence of ethanol, the ALCR protein is inactive, but when ethanol is added, ALCR and ethanol interact to form an activated ALCR, which then binds to the promoter of the target gene such as alcA, inducing the expression of the gene . This system has been successfully used to control different genes in tobacco, Arabidopsis, potato, oilseed rape, tomato, and rice . As ethanol is less expensive, readily available, non-toxic in moderate amounts and can be easily supplied to the plants, this system is considered to have a great potential for field application . In this way, the manipulation of NCED gene expression using the alcohol-inducible promoter  could be used to stimulate ABA biosynthesis and responses in plants before drought stress occurs in a commercial setting. This approach would allow the control NCED expression and subsequent ABA generation during an effective timeframe, triggering preemptive xylem development and functionality, decreasing BER incidence. Therefore, growing raspberries in pots the objective of this study was to use ALC::NCED transgenic tomato plants to manipulate NCED expression and ABA biosynthesis in order to increase xylem functionality, fruit Ca2+ uptake, as well as improve plant WUE and diminish oxidative stress responses, reducing losses due to BER incidence.

Blossom-end rot incidence was markedly different between the wild type ‘New Yorker’ and the transgenic ALC::NCED lines 1 and 2 . Wild type plants had 52.6% of BER incidence , whereas both transgenic lines had less than 20% of BER incidence . NCED gene expression was higher in the transgenic line 1 and 2, after ethanol induction compared to NY . There was no difference in xylem functionality in the proximal fruit tissue among genotypes at 15 DAP . However, xylem functionality in the distal fruit tissue was different between wild type and transgenic lines at 15 DAP . Wild type fruit had a lower density of stained xylems compared to lines 1 and 2. At 30 DAP, line 1 presented a higher number of functional xylems compared to line 2 and NY in the proximal fruit tissue. On the other hand, in the distal fruit tissue lines 1 and 2 presented higher xylem functionality compared to the wild type NY . Fruit number, fresh and dry weights; diameter and length were higher in wild type plants compared to the transgenic lines . Wild type plants had higher water loss than transgenic lines, which resulted in severe water stress symptoms, reaching almost 90% of soil water lost among the 14 days of water monitoring . Both transgenic lines showed 60% of water loss during the evaluation period . Transgenic Line 1 plants had higher relative water content than wild type and transgenic line 2 plants . There were no differences in plant dry weight among genotypes . Transgenic line 1 plants had higher WUE than transgenic line 2 and wild type plants . Ca2+ concentration in the distal fruit tissue was higher in transgenic lines 1 and 2 than wild type . Transgenic lines 1 and 2 showed an average of 12% more Ca2+ concentration in the distal fruit tissue, compared to wild type . Chlorophyll A, chlorophyll B, and carotenoid contents were lower in wild type leaves compared to transgenic lines 1 and 2 leaves . Antioxidant capacity in leaves and distal fruit tissues were lower in wild type plants, compared to transgenic line 1 and 2 plants .Abscisic acid is known to be an important signaling molecule involved in many plant responses to stress conditions . ABA biosynthesis is mainly regulated by the increase in NCED expression that catalyzes a rate-limiting step in ABA production . Indeed, many studies have shown that NCED expression is highly correlated to ABA concentrations in petunia , rubber tree , tomato , Arabidopsis thaliana , and tobacco plants .

Our study shows a potential molecular approach to manipulate the internal plant NCED expression, increasing ABA biosynthesis and leading to higher fruit xylem functionality and Ca2+ concentration in distal tissues, as well as improving plant WUE and reducing fruit susceptibility to BER. The ALC::NCED transformed plants showed a great reduction in fruit susceptibility to BER, when NCED gene expression was stimulated by ethanol treatment, compared to wild type plants. Similar ABA responses on decreasing BER incidence have been reported in other studies with external ABA spray treatments . Treating tomato plants with ABA resulted in higher number of functional xylem vessels at later stages of fruit growth and development, favoring higher xylemic water and Ca2+ uptake into the fruit. Indeed, our study has also shown that triggering NCED expression, and possibly higher ABA biosynthesis in ALC::NCED transformed plants, resulted in a higher number of functional xylem vessels at earlier stage and later stages of growth and development . The higher number of functional xylems in ALC::NCED fruit resulted in higher fruit Ca2+ uptake and translocation into distal tissues, reducing fruit susceptibility to BER, as suggested in previous studies . Some studies have shown that ABA plays an important role on increasing primary and secondary vascular bundle development . Ramachandran et al. showed that xylem vessel differentiation is increased through protoxylem in response to VASCULAR-RELATEDNAC DOMAIN transcription factors expression that are activated by ABA treatment in Arabidopsis thaliana. Molecular and genetic analyses revealed that the two ABA-mediated xylem developmental changes are regulated by distinct members of this transcription factor family, with VND2 and VND3 promoting differentiation of metaxylem cells, while VND7 promotes the conversion of metaxylem into protoxylem . Moreover, higher NCED expression in transgenic lines also resulted in different plant and fruit growth and development, compared to the wild type plants. Transgenic plants and fruit grew slower than wild types. In addition, transgenic lines had fewer flowers and more abortion that resulted in lower number of fruit per plant, compared to wild type plants. The increase in NCED expression and ABA biosynthesis have also been suggested to result in higher expression of genes coding for DELLA proteins that inhibit plant responses to gibberellins. The ABA inhibition of gibberellin responses could explain the smaller fruit observed in the transgenic tomato lines, considering that gibberellins play an important role on triggering fruit growth . Indeed, mutant genotypes with repressed DELLA expression, such as PROCERA, have bigger plant phenotypes that produce larger fruit and leaves . In addition, previous studies have shown that gibberellins increase fruit susceptibility to BER . Therefore, triggering NCED expression in ALC::NCED transformed genotypes possibly decreasing BER incidence by inhibiting plant and/or fruit responses to gibberellins.In our study, triggering NCED expression in ALC::NCED transgenic plants resulted in a reduction of plant water loss, possibly due to reduced stomatal conductance, which is one of the main plant responses to ABA . ABA plays an important role in the plant response to drought by regulating stomatal conductance and root hydraulic conductivity . When applied exogenously, ABA causes rapid stomatal closure and reduces water loss through transpiration . However, CO2 diffusion into leaves becomes limiting to photosynthesis due to stomatal closure , and this also explains the smaller fruit growth, plant pot with drainage partially caused by DELLA repression, discussed above.Besides the fact that ABA can increase WUE by changing how plants can use water to produce assimilated carbon, a new study shows that ABA over expressing tomato lines can also increase xylem embolism under water stress conditions, in response to high ABA levels in plant tissue . However, the causes for the increase vascular embolism are still unclear. Thus, increases in WUE must be seek and analyzed case by case. Our results show that treating ALC::NCED transgenic lines with ethanol might have resulted in higher ABA biosynthesis in these plants, which was not observed in the wild type plants. In addition, triggering NCED expression in ALC::NCED transgenic plants also increased leaf chlorophyll and carotenoid contents , as well as leaf and fruit antioxidant capacity. Recent studies also show that ABA can play an important role on regulating antioxidant defenses during water stress conditions .

According to Jiang and Zhang , the accumulation of ABA induced by water stress causes an increase in the levels of ROS, triggering the antioxidant defense system in plants that leads to an increase in the levels of superoxide and hydrogen peroxide radicals, which was followed by an increase in the activity of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase.In our study, the antioxidant capacity was higher in leaf and fruit tissues of ALC::NCED transgenic plants treated with ethanol, compared to the wild type. Proteomic and transcriptomic analyses performed in several plant species subjected to different stress combinations have highlighted the importance of the antioxidant defense machinery to avoid cell death and plant tissue damage . Plants with higher antioxidant capacity and/or lower ROS levels have been shown to have higher tolerance to stress conditions . Indeed, our results show that higher antioxidant capacity was highly correlated with lower fruit susceptibility to BER in ALC::NCED transgenic plants. Calcium is a nutrient that acts as structural compound, binding to pectins and improving the structure and strength of the cell wall , but it is also an essential nutrient, involved in the proper membrane stability and functionality . The symptoms of BER have been suggested to develop in response to membrane damage caused by low apoplastic soluble calcium content and/or oxidative stresses , which also corroborate with our results showing that higher fruit antioxidant capacity due to higher NCED expression inhibited BER symptoms development possibly by inhibiting membrane damage caused by high ROS levels.Blossom-end rot is a physiological disorder believed to be regulated by the interaction between the genotype and environmental conditions. Under stress, such as drought, heat, salinity, and others, each genotype must have the capacity to trigger morphological, biochemical, physiological, and molecular changes that will maintain proper cell and plant tissue metabolism, avoiding cell death and tissue damage. According to our study, triggering NCED expression in ALC::NCED transgenic plants resulted in higher ABA biosynthesis that is supported by the observed lower plant water loss, leading to morphological changes such as lower fruit number, size, weight, and higher functional xylem vessels in the fruit, as well as biochemical changes such as increased chlorophyll and carotenoids contents and antioxidant capacity in leaves and fruit, and physiological changes such as reduced leaf transpiration and improved plant WUE, and possibly molecular changes related to the communication among ABA and other plant hormones that resulted in tomato plant with better performance under stress, using less water to produce fruit with less BER. The possibility to control plant ABA biosynthesis, using the ALC:: NCED approach, could enable the maintenance of higher plant ABA levels, promoting a reduction of cultivation costs in regions where irrigation water is limited, as well as turning well irrigated region in more efficient crop production sites. Besides that, this approach could help studies in which ABA is necessary only during certain parts of plant growth and development, in order to avoid negative effects of higher ABA levels, like short size, smaller fruits, floral abortion or even during a specific timing, when it is necessary to maintain the stomata close. In addition, the crosstalk between plant hormones could also be studied since ABA production can be easily manipulated. Therefore, this technique represents a powerful tool to improve plant WUE and allow for Ca2+ to reach the distal end of the fruit, decreasing BER incidence and reducing fruit losses. In addition, more studies must also be conducted, to check if ABA could lead to a higher xylem density and maintenance in fruit tissues, if the VND transcription factor family were highly expressed, and finally if genes related to GA-repressed growth were expressed in response to ABA treatment.

Unlike ACS and ACO1, SlCBF1 showed a clear spatial differentiation in both chilling and control conditions. Expression was higher in the pericarp compared to the columella , which suggests that the former might be more responsive to cold stress, possibly due to its external localization. Under chilling in both tissues, SlCBF1 expression peaked at 1h and was sustained for 24h in our study, which can be described as an early response , it then declined to the levels observed at the control temperature, after 3 weeks cold treatment. In other studies, cold storage induced expression of SlCBF1 for up to 8h; 8 days, and 14 days, however there was no induction at 6 °C in Micro-Tom fruit. Te upregulation of SlCBF1 may therefore be dependent on fruit developmental stage, the severity of cold stress, and genotype. After rewarming of the ‘control fruit’, SlCBF1 expression increased to levels higher than during chilling . This may be suggestive of two things: first, that SlCBF1 transcripts in chilled tissues were unable to reach the same levels as tissue held in control temperature, since they were developmentally repressed; and second, SlCBF1 is involved in ripening independent of ACO1 or ACS2, given the asynchrony of their expression . However, a correlation between endogenous ethylene production and SlCBF1 upregulation could partly explain this behavior and matches our observations . Dehydration stress response. Dehydrins , are protective proteins that accumulate in response to dehydration-associated stresses, including chilling. The expression of the clone FC11CA08-2, here named DHN, was analyzed in this study. DHN mRNA levels increased only after 24 h and 3w of post harvest chilling inthe pericarp, drainage collection pot or 3w in the columella with decreases in both tissues after rewarming. There were no detectable differences between tissues, however they responded differentially to temperature .

After rewarming, ‘control pericarp’ DHN expression was higher than that in the ‘chilled pericarp’, due to ripening taking place in the control. It appears that DHN transcript abundance in fruit increases as ripening progresses. The magnitude of changes were greater at 2.5 °C compared to 12.5 °C, consistent with a higher requirement for the molecular chaperones encoded under cold-stress. Oxidative damage. Prolonged or intense chilling stress induces ROS overproduction, which accelerates cell death. The alternative oxidase pathway is activated to minimize ROS levels, and in tomato fruit, AOX1a has been associated with enhanced PCI tolerance. Therefore, AOX1a was studied here. Lipoxygenases catalyze the peroxidation of polyunsaturated fatty acids and are associated with both ripening and redox balance, processes affected by PCI. The expression of the LoxB isoform has not been studied during fruit post harvest chilling storage, and was included. AOX1a expression levels in pericarp and columella were similar, but chilling induced a differential response over time. Transcript levels in the pericarp peaked at 24h and 3w, similar to that seen by Fung et al.. The ‘chilled pericarp’ had a reduced AOX1a expression after rewarming while the ‘chilled columella’ changed little even after rewarming. Under control conditions in both tissues, gradual increases were observed, but rewarming enhanced AOX1a expression , matching ethylene production rates , consistent with ethylene regulation of this gene. ACO1, ACS2 and AOX1a, were co-expressed, but chilling suppressed this correlation . PCI therefore contributes to the uncoupling of ripening-related ethylene biosynthesis, highlighted by the inability of chilled tomato to resume normal ripening after rewarming . LoxB expression displayed a mixed spatial-response that varied with temperature. Expression in both pericarp and columella was unchanged at 1h and 24h .

After 3 weeks, expression was down regulated, but in contrast, rewarming induced the upregulation of LoxB in both tissues. LoxB expression matched ethylene production, consistent with its regulation by this hormone. LoxB expression also paralleled MDA values after rewarming in the columella , in agreement with membrane alterations induced by PCI. The correlation of LoxB with ethylene production rates and ripening was in accordance with the strong correlation between LoxB and ACS2 at 12.5 °C . Interestingly, transcript levels in the ‘control pericarp’ plus rewarming were higher than those of rewarmed tissue after chilling, even though ethylene levels were 1.2-fold higher in the latter. In this case, ethylene production increased in response to chilling-induced stress and not due to ongoing ripening.Principal Component Analysis was performed to explore the structure of the gene expression data from a spatial perspective with respect to cold storage and rewarming of chilled tissue . The first and second principal components explained 75 and 15% of the variation present in the data, respectively. Data for the pericarp and columella portions under chilling for 3 weeks separated from the rewarmed tissues. More importantly, the data distinguished among tissues, with the pericarp and columella showing a clear separation even though gene expression differences between cold and rewarming were a greater determinant of the patterns seen on the PCA. Overall this analysis supports the hypothesis of a spatial and temporal differentiation in response to chilling stress at the gene expression level.Post harvest chilling injury is a complex multifactorial disorder with detrimental effects on tomato fruit quality and shelf-life. With the aim of representing the tomato fruit as a multilayered and integrated system of response to cold stress, we analyzed PCI impact on different fruit tissues and correlated it with known physiological parameters .

Overall, cold stress uncoupled key molecular, biochemical, and physiological processes occurring during the normal progression of storage and ripening. Increased water mobility and tissue liquefaction were also disrupted as evidenced by MRI-obtained D-values from the pericarp, columella and locular portions, and ion leakage obtained from the pericarp. MRI and color development confirmed three concepts: first, the system’s inability to restore or repair the chilling-affected mechanisms; second, that PCI is cumulative and progressive over time; and third, the need to examine each tissue to characterize PCI’s progression and symptomatology, since the most studied fraction, the pericarp, may not reflect processes occurring in other tissues. Reduced starch breakdown in columella and seed discoloration during cold storage reflect that, besides external changes, PCI extends to internal tissues. Tissues exhibited heterogeneous patterns of response to PCI at the biophysical, biochemical and molecular levels. D-values were intrinsically different in the three tissues under study, and their time evolution and temperature responses were also mixed. Responses to oxidative damage represented by the lipid peroxidation byproduct MDA varied in response to temperature but peaked after rewarming, which again highlights that after crossing a threshold of cumulative cold damage, rewarming aggravates PCI’s manifestation instead of alleviating it. Starch accumulation also showed significant spatial differences, suggesting that tissues may display a sharper specialization at the metabolite than at other levels. Responses to cold from the perspective of gene expression were highly dependent on the tissue-type, temperature and time of storage, but overall, they paralleled ethylene production trends via stress response or ripening. Some genes seemed to act concertedly across experimental conditions , others acted coordinately under either cold or control conditions or under apparently independent programs . Transcript accumulation was higher in the pericarp across conditions , square plastic pot equally expressed in both tissues , or, dependent on temperature and storage time . Taken together, this evidence reveals the dynamism of cold-stress in the tomato system and suggests that fruit may display specialized mechanisms to elaborate a response to this environmental challenge. It also unfolds numerous questions about the nature of such varied responses among fruit tissues: are they advantageous to the fruit under stress? What is the source of these differences? Would such relationships differ in the fruit from cold-tolerant tomato species? Exploring these questions in a comprehensive way may deepen our knowledge of this complex phenomenon to elaborate long-term, robust solutions.The efficacy and palatability of commercially available rodenticides can vary greatly, and bait effectiveness is often specific to particular pest species . Many rodenticides have been developed to control rodent populations , and several studies have assessed the materials’ ability to control rats and mice in natural areas . However, until now no peer-reviewed studies had tested the efficacy of rodenticides for roof rat control in nut or tree fruit crops, and few if any studies had been conducted on deer mice. We recently initiated an investigation into the efficacy of three rodenticide baits for control of roof rat and deer mouse activity in almond orchards and found that the 0.005% diphacinone oat bait, sold in many county Agricultural Commissioner’s offices, was highly effective . This study made use of elevated bait stations, which proved effective at supplying bait to target species while substantially limiting access to rodenticides for many non-target species. In this publication we provide information on how to identify damage from roof rats and deer mice in nut and tree fruit orchards, and how to effectively implement a baiting program to control these pests. This appears to be an efficacious, cost-effective, and safe baiting protocol for control of roof rats and deer mice in orchard crops, something that has thus far been unavailable to growers.Accurate identification of the species responsible for damage is essential to development of an effective pest management program. If your management plan focuses on the wrong species, it is likely to be ineffective and it may pose hazards to non-target species and even be an illegal misuse of the material, based on the rodenticide label information.

Fortunately, the presence of roof rats and deer mice can often be detected through indirect monitoring techniques. For example, roof rats often burrow at the bottom of trees, and these burrows are typically 2 to 3 inches in diameter . Burrows of the California ground squirrel are sometimes this same size, but usually they are a bit larger . Also, if ground squirrels are present, you will see them running around above ground and hanging out in burrows.Discarded almond shells at the entrance of a burrow can help you determine the depredating species , but distinguishing between damage from deer mice and roof rats can be difficult. Deer mouse burrow openings typically average around 1.5 inches in diameter. If burrow openings of this size are present, the depredating species may be the deer mouse. Vole and deer mouse burrow openings are similar in size, but voles are not typically found in almond orchards, so long as ground cover is limited. If burrow openings are larger , the roof rat is the likely culprit.he bait stations used in our field trials were tubular structures manufactured specifically for Orange County Vector Control . The bait station consisted of high-density polyethylene plastic tubes that were 13 in long and 3.94 in inside diameter . A steel end cap was fixed onto each end of the tube. Each end cap was penetrated with a 1.89-in opening, big enough to allow the roof rats and deer mice to enter the station and small enough to reduce or even eliminate any inadvertent loss of bait from the bait station. On the inside of the metal cap, under the opening, a 4.5-in long metal shelf is present. This also helps reduce bait loss. As of this writing, these bait stations are available for sale in a limited supply from the Los Angeles County Agricultural Commissioner’s office. We are exploring additional supply options.The current label for 0.005% diphacinone oat bait only allows baiting during the non-bearing season. This means that growers need to be proactive when dealing with rodent infestations. It is the responsibility of the grower to be aware of the presence of endangered species in orchards where they intend to implement a control program, since the bait may prove hazardous to non-target species. The killing of an endangered species may result in a fine and imprisonment under the Endangered Species Act 1973. The use of elevated bait stations will eliminate access to bait for many protected mammal species, such as kangaroo rats . Although other protected species, such as the Tulare grasshopper mouse , are not usually associated with climbing trees, growers must be vigilant in areas where these and other protected species are found. Growers can consult the California Department of Pesticide Regulations PRESCRIBE website for any endangered species restrictions associated with bait application. We recommend placing bait stations either 98 feet or 164 feet from each other, throughout the orchard.

However, some pathogens can exploit the SA/JA antagonism for their own benefit ; for example, B. cinerea produces an elicitor ofSA responses through the NPR1-dependent pathway, which leads to the inactivation of two JA-response genes, Proteinase I and II, that are required for resistance against necrotrophs . ET can counteract the negative effects of NPR1 on JA responses, but it also enhances the NPR1-dependent expression of SA defense genes . Leon-Reyes et al. proposed that the concurrent activation of ET and JA pathways promotes plant insensitivity to subsequent SA-mediated suppression of JAdependent defenses, which then favors effective resistance against pathogens of different lifestyles. Hence, localized synthesis and perception of JA, ET, and SA at the appropriate relative concentration and timing appear to be required for plant resistance. During infections of fruit, ET, SA, and JA networks might interact to stimulate defenses. Nonetheless, accumulation of susceptibility factors as a consequence of ET-triggered senescence/ripening and the antagonism between SA and JA responses may represent opposing influences in the fruit–pathogen interaction and, thus, lead to susceptibility.Increased expression of the tomato 9-cis-epoxycarotenoid dioxygenase 1 , a key ABA bio-synthetic gene, occurs during early infection of susceptible fruit , which suggests a link between ABA synthesis and fruit susceptibility. Several plant pathogens, including B. cinerea, generate ABA during infection or use effectors to induce its production by the host, round plastic plant pot facilitating senescence/ripening and subsequent colonization of the ripened tissue . ABA has been involved in fruit ripening of climacteric and non-climacteric fruit .

Exogenous treatments of ABA induce the expression of the ripening-associated ET biosynthetic genes LeACS2, LeACS4, and LeACO1, thereby, triggering ET production and ripening . In tomato fruit, expression of the 9-cis-epoxycarotenoid dioxygenase 1 increases at the onset of ripening prior to the ET climacteric rise . A slight induction of LeNCED1 was detected in infected MG fruit , which could have been prematurely induced to initiate climacteric ripening; however, a significant decrease in expression occurs at the late stage of ripening . The development and analysis of a genetic knock-out mutant line in LeNCED1 will be instrumental to understand the impact of ABA synthesis during the increase in ripe fruit susceptibility. The expression of FLACCA, a tomato molybdenum cofactor synthase that is involved in ABA biosynthesis, increases as consequence of ripening, but it is reduced in response to the B. cinerea infection . These observations indicate that the plant may reduce the expression of FLACCA in an effort to contain the rise in ABA production caused by the pathogen colonization; however, experimental evidence is needed to test this hypothesis. The interaction between tomato fruit and B. cinerea results in significant changes in the expression of 37% genes involved in the ABA signaling pathway . Alterations in regulators of ABA signaling/responses are detected as well as changes in membrane protein channels . In general, increased expression of the PYL/PYR/RCAR receptors was observed in RR fruit . The PYL/PYR/RCAR receptors are positive regulators of ABA response by blocking the PP2Cs inhibitors . In Arabidopsis, suppression of three PP2C clade A phosphatases results in constitutive activation of ABA signaling and increased susceptibility to fungal infection .

In agreement with these results, significant up-regulation of a RCAR1 homolog and down-regulation of a PP2C homolog in infected RR fruit at 1 and 3 dpi provides further support for a positive relationship between ABA responses and susceptibility . Enhanced expression of suppressor genes throughout the ABA hormonesignaling network is detected after inoculation with B. cinerea of resistant MG fruit . In contrast to the increased expression in MG fruit, the homolog RACK1_a is significantly down-regulated in RR fruit at 1 and 3 dpi . Previous studies have demonstrated a role for RACK1 in the activation of defense mechanisms in response to pathogens in rice. The rice RACK1 homolog triggers ROS production, defense gene expression, and disease resistance by interacting with OsRac1, a Rac/Rop small GTPase involved in basal immune responses . It is plausible that tomato homolog of RACK1 has a similar role in fruit by controlling infections in MG fruit. The contribution of ABA to the enhanced susceptibility of ripe fruit is supported by the disease development assays with the tomato sitiens mutant which fails to synthesize ABA . Inoculation of RR sitiens fruit with B. cinerea resulted in a significant decrease in disease incidence when compared to the infected wild-type RR fruit . Interestingly, about 40% of the inoculated sites in RR sitiens fruit displayed the typical localized necrotic response of wild-type MG green fruit . MG sitiens fruit are as resistant as MG wild-type fruit . The molecular mechanisms that mediate the reduction of susceptibility in RR sitiens fruit are not known; however, analysis of necrotrophic infections in leaves of sitiens plants suggest that a strong induction of defense-related genes , the oxidative burst, and an increase in cuticle permeability might be crucial for the resistant phenotype of this mutant .Plants modulate the ET, SA, JA, and ABA hormone networks to induce immune responses against the attacks by various classes of pathogens .

Recent studies indicate that other hormones such as auxin, gibberellins, cytokinins, cell wall oligogalacturonides, and brassinosteroids might also be implicated in responses to pathogens either directly or by interacting with other hormones . The interactions among hormones provide the plant with a powerful regulatory potential, but also give opportunities for pathogens tomanipulate the plant defense-signaling networks to their advantage . Plants in their natural environments infrequently interact with a single pathogen species, rather they are impacted by microbial communities, herbivores, and other plants, all of which could individually, collectively or cooperatively influence responses to contact with pathogens. This complexity should be taken into account when studying plant–pathogen associations. In fruit, high levels of ET and ABA, which stimulate senescence/ripening processes, may facilitate colonization by necrotrophs. The balance between SA and JA responses seems to be crucial for resistance in unripe fruit, while ABA production correlates with ripe fruit susceptibility. ET, at appropriate concentrations, also contributes to the resistance of fruit by activating JA and/or ET responses and possibly by blocking the antagonistic effect of SA on JA signaling. Hence, the role of plant hormones in promoting fruit resistance or susceptibility depends on the interaction of several factors, including: the concentration of the hormones, the timing of the synthesis and perception of the hormones, the competence of the host tissue to respond to active forms of the hormones, the localization of the plant’s response to the hormones, and the pathogen’s infection strategy, including its own production of hormones. The interaction between tomato fruit and B. cinerea causes transcriptional reprograming of multiple plant hormone networks simultaneously, and, depending on the developmental stage of the fruit contributes to either resistance or susceptibility outcomes. In Figure 6, we provide an overview of key expression changes of genes involved in biosynthesis, modification, signaling, and response pathways of the hormones that, based on our transcriptome profiling analysis and validation, we propose to be part of the regulation of the resistance-to-susceptibility transition associated with ripening and healthy fruit ripening. Analytical methods that allow the simultaneous profiling of multiple signaling molecules that are produced during fruit infections , will shed further light on the signaling networks that control fruit susceptibility in the context of ripening, but the challenge of identifying whether the hormones are synthesized by the host or by the pathogens will still be a limitation. New strategies to study complex gene networks involved in hormone signaling in fruit–pathogen interactions, including the analysis of natural or induced mutants in both plants and pathogens, 25 liter round pot the use of laser micro-dissection and cell-specific transcriptomics, and metabolomics can contribute novel important information to our understanding of the biological and ecological importance of plant development in modulating resistance and susceptibility. From an applied perspective, evaluating the specific hormonal events that promote fruit susceptibility may facilitate the development of commodities that ripen successfully and yet are less susceptible to pathogen infection.Diabetes is a multifaceted metabolic disorder affecting carbohydrate, fat, and protein metabolism. It is caused by increased levels of circulating blood sugar and insulin deficiency characterized by abnormal insulin secretion and insulin resistance in the body. Type-2diabetes affects 80% of those living with diabetes and is largely due to an unhealthy diet and a sedentary lifestyle, which might lead to persistent high glucose levels in the blood, oxidative stress, aging, and other metabolic imbalances. The use of the non-vertebrate organism Drosophila melanogaster as a model tool for research on various human diseases is important because this fly has biochemical features and characteristics similar to those of mammals; therefore, its use is increasing.

This model organism has been tested and has gained worldwide reputation for use in biomedical research, such as in diabetes studies and in other studies involving genetic manipulations. Certain reports identified that disease-causing genes in humans are conserved in Drosophila melanogaster, such as those associated with the insulin-like growth factor signaling pathway. About 70–75% of the brain cells in the fruit fly are insulin producing cells that are similar to the vertebrate pancreatic β cells and secrete seven insulin-like peptides. In addition to this, using the fruit fly for research studies is easy and cost-effective, since it is not expensive to maintain them. The indigenous eggplant belongs to the Solanaceae family and is locally called “Igba Yinrin” by the Yoruba people in South-West Nigeria. It is commonly known as a forest bitter berry and a non-tuberous and highly polymorphic indigenous medicinal fruit that is widely distributed in non-arid areas of Africa and has nutritional and therapeutic potential. A study reported that ripe and the unripe eggplants are used in folklore medicine. These eggplants is also used in Ghana and Cameroon cuisine as one of the ingredients of a dish called “Nkwi”. Despite the benefits of the unripe fruits being known, no study has reported the properties of the ripe fruit which is usually left to waste and rot in farms. The ripe eggplant fruits are usually discarded partly due to their low acceptability and the ignorance of their benefits and thus are among the post-harvest food crops lost after harvest in Nigeria, West Africa. Meanwhile, a study showed that ripening brings changes in the fruit content of phenolic compounds, organic acids, and carbohydrates, as well as in its color, texture, and flavor. Nigeria is facing an immense food insecurity challenge, a problem partly attributed to the increasing loss of food crops during post-harvest handling and distribution to retail markets. The reduction in post-harvest loss of this eggplant fruit and its use as a food ingredient for functional food development, such as in cookies and dumplings, could promote sustainable food security and a better health treatment/management of noncommunicable diseases and cardiovascular diseases , a task that requires an integrated approach . Therefore, this research was carried out on both ripe and unripe eggplants to compare their biological effects, with a focus on the anti-inflammatory, antioxidant, and anti-diabetic properties of a diet containing ripe Solanum anguivi lam fruits in diabetic Drosophila melanogaster .Ripe and unripe eggplant fruits were obtained from the botanical garden of the FUTA, Akure, as shown in Figure S1 . The identification and authentication of the fruits were performed at the FUTA Herbarium; and were recorded with number 0291a and 0291b. Common fruit processing methods were adopted, including cleaning, sorting, cutting, pulping, drying, and grinding the fruits as needed. The fruits were processed according to the method described by.Mineral elements were analyzed according to AOAC and Perkin-Elmer. The method described in was used to determine the total carotenoid content, while vitamin C determination was carried using a method reported in [16], vitamin A content was determined according to [17], and vitamin E content was obtained according to [18]. HPLC–UV [high-performance liquid chromatography with ultraviolet detection] was carried out on the samples as described in [19]. This is an advanced technique used for separating and quantifying bio-active compounds in a mixture. In this method, a liquid sample is loaded in a column, and its components are detected by their absorption of ultraviolet light, which provides quantitative and qualitative information. The standard compounds in Table 1 were investigated and identified according to a standard protocol.The eggplant fruits were washed, cut, and then blended into a puree. The puree was filtered through cheesecloth to collect the liquid, which was the eggplant extract and was freeze-dried.

As part of our horticultural program, we have crossed various cultivars of D. kaki and obtained their progenies segregating for fruit astringency. One of these crosses was made between two genetically distinct NA-types of D. kaki, cv. Luo Tian Tian Shi and cv. Taishu. Their F1 progenies were segregated into A- and NA-types, the latter of which appears to be determined by the presence of a dominant allele derived from cv. Luo Tian Tian Shi . In this report, we made use of these materials, attempting to elucidate the molecular mechanism of soluble PA accumulation in young persimmon fruits. We employed suppression subtractive hybridization to identify transcripts whose accumulation patterns were distinct between the segregated A- and NA-type fruits when their PA contents became distinct. Interestingly, only a few clones encoding Xavonoid biosynthetic enzymes were identified. Among cDNAs represented by multiple clones were those encoding a new member of the 1-Cys peroxiredoxin family and that of subgroup F of family 1 glycosyltransferases . A detailed sequence comparison and phylogenetic analysis revealed that the isolated 1-Cys Prx may be a novel type. In addition, UDP-galactose:anthocyanidin/Xavonol 3-O-galactosyltransferase activity of the GT homolog was conWrmed by using a bacterial expression system. These results may suggest complex mechanisms of PA accumulation in persimmon fruits.Generation of F1 progenies between the two distinct NAtype persimmon cultivars , their growth, and collection of their fruits were partly described by Ikegami et al. . Fruits usually become fully mature and ready to harvest for human consumption in the end of October to November. Immature green fruits used in this study were harvested considerably earlier, at three different dates , 25 liter plant pot during which PA accumulation started to decline in the NA-type but not in the A-type . The A- and NA-types used in this study were defined previously based on the size of PA-accumulating cells and the soluble tannin content of fully mature fruits . Fruit fresh was separated from seeds and peel, cut into small pieces , frozen with liquid nitrogen, and stored at ¡80°C before further analysis.

Total RNA was isolated from 5 g of the frozen sample using the hot borate method .Amino acid sequences were aligned with ClustalX . Phylogenetic analyses of the aligned amino acid sequences based on maximum parsimony were implemented in PAUP* with heuristic searches using the TBR branch-swapping algorithm 1,000 random taxon addition replicates and no limit on the number of trees saved. Relative support for clades was assessed using 1,000 bootstrap replicates with 10 random taxon addition replicates per bootstrap replicate. For the Prx sequences, 191 ambiguously aligned sites were excluded from the original alignment of 357 sites. A Wnal data set of 166 characters, of which 3 were constant, 6 were variable but parsimony-uninformative, and 157 were parsimony-informative, was subjected tophylogenetic analysis. The tree shown in Fig. 4a is one of four most parsimonious trees , arbitrarily rooted along a strongly supported internal branch. For the GT sequences, the complete alignment of 610 amino acid characters, of which 61 characters were constant, 62 were variable but parsimony-uninformative, and 487 were parsimony-informative, was used in phylogenetic analysis. The unrooted phylogram shown in Fig. 5a is one of three most parsimonious trees .The astringency of A-type fruits and that of NA-type fruits became distinct in those harvested in August. That is, the soluble PA content per dry weight of A-type fruits remained almost constant from June to August, whereas that of NA-type fruits dropped by more than 50% . By contrast, during this period, the concentrations of insoluble PAs remained largely unchanged and at comparable levels between the two fruit types ; and fresh weight of a fruit of both types increased similarly up to five times . These data suggest constant PA biosynthesis in A-type, but not in NA-type fruits.

It may also be possible that the level of Xavonoid oxidation, which has been shown to be negatively correlated with PA solubility , was lower in A-type than in NA-type. Hence, we decided to compare the transcript accumulation patterns in NA- and A-type fruits harvested in July and August. To identify differentially accumulating transcripts, we used suppression subtractive hybridization , which recently allowed us to isolate several transcripts including those encoding PA-biosynthetic enzymes whose accumulation levels were up- or down-regulated in persimmon fruits by ethanol treatment to remove astringency . We prepared RNAs from A- and NA-type fruits and generated reciprocal cDNA SSH libraries for samples of each data point. Among 4,800 recombinant clones that were randomly selected from these libraries, 37 clones showed significantly different accumulation patterns between the two fruit types by RNA dot blot assays, and their nucleotide sequences were determined . Based on the previous results , we had expected to find a number of PA-biosynthetic genes in A–NA libraries. Indeed, a total of nine independent clones involved in phenylpropanoid metabolism were isolated . However, none of them encodes an enzyme catalyzing one of the committed steps for PA biosynthesis, i.e., LAR or ANR. Some other clones identified in these libraries encode homologs to known proteins that do not play a direct role for PA biosynthesis . We also attempted to obtain full-length coding sequences of SSH clones isolated from A–NA libraries in order to facilitate further analysis. To this end, we screened a cDNA library from A-type persimmon fruits, and were able to obtain apparent full-length sequences for a subset of cDNAs . The presence of clones for the three Xavonoid biosynthetic enzymes, phenylalanine ammonia lyase , chalcone synthase , and dihydroXavonol 4-reductase , in A–NA libraries was consistent with the preliminary RNA-blotting data . Also identified in A-NA libraries were clones for two other Xavonoid biosynthetic enzymes, cinnamic acid 4-hydroxylase and Xavonoid 3 5 -hydroxylase .

Three truncated sequences may derive from nonoverlapping portions of a single mRNA encoding a C4H homolog . In addition, three SSH clones from the July sample were found to encode 3-dehydroquinate dehydratase/shikimate 5-dehydrogenase , which is involved in the biosynthesis of aromatic amino acids to fuel the phenylpropanoid pathway. Finally, represented by the highest number of clones for both the July and August samples were the transcripts encoding a protein with high sequence identities to various plant Xavonoid GTs, such as UDPGlc:anthocyanidin 3-O-GlcT from Xower buds of Lobelia erinus L. , anthocyanin 3-O-GalT from cell suspension culture of Aralia cordata Thunb. , and kaempferol 3-O-GalT from pollen of Petunia x hybrida . The group of cDNAs not directly involved in phenylpropanoid metabolism consists of a total of eight independent clones. Three of them were found in A–NA libraries, encoding proteins similar to glucose acyltransferase , 1-Cysperoxiredoxin , and glutathione S-transferase , respectively. SCPL might be involved in PA accumulation, since correlation of its gene expression and accumulation of PAs has been reported at least twice, one in persimmon fruits and another in hairy roots of grape overproducing MYB transcription factors . The clone encoding a 1-Cys Prx homolog was represented by five SSH clones, whereas GST, which was shown to have multiple functions including conjugation to anthocyanidins in the cytoplasm for vacuole sorting , was represented by a single clone . Among the five cDNAs found in NA–A library, three clones may encode non-overlapping portions of a single protein with a high sequence similarity to LATE BLOOMER 1 from pea, which plays roles in photoperiodic Xowering, de-etiolation, and circadian regulation . The other two appear to encode distinct chitinases, among which is one for a class II chitinase-like protein, black plastic plant pots which was represented by Wve SSH clones.The citrus fruit, termed hesperidium, is a fleshy fruit which, like all berry-type fruit, is characterized by a thick and fleshy pericarp . The pericarp is usually divided into three tissues: the exocarp, which is the outer skin, the mesocarp, which usually refers to the major fleshy, edible interior, and the endocarp, an internal tissue composed of one or several cell layers. In true fruit, which develop from the ovary, these three tissues are part of the ovary wall. The exocarp of citrus fruit is the outer colored peel, often referred to as the flavedo . Proceeding inward is the albedo, the spongy white part of the peel. Most cell layers of the albedo are considered to be mesocarpal tissue, and the two or three innermost cell layers are referred to as endocarp . In mandarins, the albedo disintegrates during fruit maturation, leaving only the vascular system , which gives this group its name, Citrus reticulata. The pulp, the edible part of the fruit, is composed of juice sacs/vesicles that develop from the endocarp at an early stage of fruit development . Some authors refer to the juice sacs as endocarp, while others consider them to be a separate tissue. The juice sacs develop into the ovary locule, defined as the section in which the ovary wall that develops into fruit. The carpel and the juice sacs are covered by the same epidermal layer of segment epidermis . The juice sac is connected to the wall by a stalk, which joins the segment epidermis, so the latter provides one continuous layer covering both the segment and the juice sac.

Three major vascular bundles, a dorsal and two side bundles, are found in each section. Most juice sacs initiate from the dorsal wall, but some develop from the side wall, adjacent to the side vascular bundle . When present, seeds develop in the inner side of the fruit, where the carpels merge or along the ovary wall. Nutrition is supplied by a specific bundle, termed seed bundle, reaching from the fruit pedicle to the center of the fruit.The juice sac is a unique structure, found only in fruit of the genus Citrus and its close relatives. It is often referred to as a “sac of juice,” but this is misleading; the juice sac is composed of various layers of cells, each with distinct morphology . The vesicle primordia emerge from the endocarp soon after fertilization and fruit set. In a few cases, juice sac primordia are visible even before fertilization and fruit set, mainly when fertilization does not occur and parthenocarpic fruit develop . During fruit development, the vacuole of the juice sac cell becomes greatly enlarged, occupying over 90% of the total cell volume, and releases its content as juice. At fruit maturity, the vacuole contains about 100, 75, and 90% of the total cellular sucrose, hexose and citrate, respectively . The juice sac is considered the major fruit sink; however, it is disconnected from the vascular system, which ends in the albedo . This unique characteristic determines photo assimilate translocation rate into the sink cells and therefore, rate of fruit development, and the time required to reach maturity.In many citrus cultivars, the major external change that marks the conversion of the citrus ovary into a fruit let is usually petal fall . Fruit development is divided into three overlapping stages: cell division , cell expansion , and fruit maturation . During stage I, fruit growth is relatively moderate, and the peel, especially the albedo, thickens by cell division. During this stage, juice sacs grow out via cell division into the locule. Stage II is characterized by rapid fruit growth, mostly due to juice cell expansion. During stage III, the rate of fruit volume increase is greatly reduced. Externally, the major change is color break, and internally, sugar and acid levels reach the desired levels for harvesting and consumption, as discussed further by Spiegel-Roy and Goldschmidt . Changes in secondary metabolites give the fruit its unique aroma and flavor . As there is no respiration burst or autocatalytic ethylene production, the citrus fruit does not undergo the classical ripening process, typical of climacteric fruits. For a given citrus cultivar, the final flavor quality of the fruit has to be determined empirically and depends, largely, on consumer preference . The completion of fruit development is cultivar-dependent, with some cultivars, such as Satsuma mandarin , being ready for harvest 5–6 months after flowering, whereas others, such as Valencia orange , are harvested 12–14 months after flowering . In hot climates, fruit development is accelerated, potentially reducing the time needed for fruit maturation by ca. 50% .