A single gene from Streptosolen grouped sister to the FUL1 and FUL2 clades, while a gene from Schizanthus, one of the earliest diverging genera , grouped as sister to the euFULII clade. To confirm the above were not artifacts, we re-assembled the Streptosolen transcriptome while searching for reads supporting the gene contig, and amplified the Schizanthus sequence using gene-specific primers. The presence of both FUL1 and FUL2 genes in species from across the phylogeny is consistent with the event that produced these two clades being part of a family-wide, whole genome duplication or triplication . However, we did not find a FUL2 ortholog in Schizanthus, using transcriptome data, or Goetzia, using PCR. These two genera are among the earliest diverging in the family , and are the earliest that we sampled. This raises the possibility that the FUL1/FUL2 clades resulted from a duplication that occurred following the diversification of Schizanthus and Goetzia. In addition, although we obtained MBP10 sequences from Nicotiana and most of the genera that diversified subsequently , we did not find members of the MBP10 clade in genera that diverged prior to Brunfelsia. This suggests that the MBP10 and MBP20 subclades were produced by a duplication that occurred later in Solanaceae diversification, after the euFULI duplication and any proposed family-wide whole-genome events.To investigate the nature of the MBP10/MBP20 duplication, we mapped the location of the four euFUL paralogs to the genome of cultivated tomato. FUL1 and FUL2 are located on chromosomes 6 and 3, respectively, hydroponic net pots consistent with their origin from a whole genome multiplication. By contrast, MBP10 and MBP20 are both located on chromosome 2, about 14.3 million base pairs apart .

The location of both euFULII genes on the same chromosome, and the presence of only one ortholog in early diverging species, support the hypothesis that these paralogs may be the result of a tandem gene duplication. Moreover, comparing a 1-million-base-pair region surrounding both MBP10 and MBP20 shows synteny, further supporting a tandem duplication . Annotations indicate that these syntenic zones contain 17 homologous regions. The regions that show homology are located on the opposite sides of MBP10 and MBP20, suggesting an inversion of the tandemly duplicated region. Although we recovered an MBP10-clade member in Brunfelsia australis using transcriptome analysis, we were unable to amplify this gene from leaf or floral tissue of Fabiana or Plowmania, genera that are most closely related to Brunfelsia . In addition, Petunia is also a member of the clade that includes Brunfelsia, and searches of the published Petunia genomes also failed to turn up an MBP10-clade member. However, the Brunfelsia sequence in our analysis, obtained from transcriptome data, falls in the expected place in the phylogeny, and we confirmed the presence of MBP10 transcript in Brunfelsia floribunda floral RNA. This suggests that the MBP10/MBP20 duplication occurred before the divergence of the Brunfelsia/Fabiana/Petunia/Plowmania clade but the MBP10 paralog was lost in Fabiana, Petunia and Plowmania.A long first intron ranging from 1 to 10 kb, with multiple potential TF binding sites, is a general feature of FUL homologs . By contrast, MBP10 has a short first intron of about 80 bp in both cultivated tomato and its closest wild relative, S. pimpinellifolium, and about 110 bp in Nicotiana obtusifolia . The expression of most euFUL genes is strong across nearly all vegetative and reproductive organs ; however, diverse analyses using both quantitative and non-quantitative methods indicate that MBP10 expression is relatively weak in tomato, S. pimpinellifolium, and N. obtusifolia in most organs , however, some studies have suggested moderate expression in leaves . To determine if the short first intron lacks putative TF binding sites, we searched the first intron of MBP10 and MBP20 in tomato .

We found that the first intron of MBP10 contains no putative TF binding sites, while that of MBP20 contains 88 putative TF binding sites for eight different TFs. These TFs belong to five main families : MYB , HSF , Dof , WRKY and MADS-box . A similar situation was observed for Nicotiana obtusifolia, which had 133 putative binding sites in thefirst intron of MBP20 for a similar array of TFs, while MBP10 had only four such sites. In addition, we searched the first intron of AGL79, the euFULII paralog of FUL in A. thaliana, and found 49 putative binding sites, also for similar TFs and TF families. This suggests a loss of regulatory motifs in MBP10.This region corresponds to the K domain . In comparison, the M and the I domains had relatively few sites undergoing diversifying selection. Since these TFs function in complexes with other MADS-domain proteins as well as other proteins, novel interactions made possible by amino acid changes in this region might lead to changes in transcriptional activity. The K domain had 14 sites undergoing diversifying selection in the FUL1 proteins and four of those showed a change in polarity . Of those four, a site that corresponds to the 153rd residue in the tomato protein had negatively charged glutamate in most of the nonSolanoideae species while all Solanoideae species had a nonpolar residue: valine or methionine . All other changes in FUL1 proteins that result in a change in charge appeared to be reversible, and none were correlated with the phylogeny nor with phenotypic changes. We used the PROVEAN tool on all four K-domain sites that showed a change in charge to predict whether these transitions were likely to be deleterious or neutral . Two of these sites, one with a histidine to glutamine/asparagine shift at the 95th residue, and one with a lysine to glutamine/threonine shift at the 157th residue , were predicted to be functionally deleterious while the other two sites, including the 153rd residue with E to V change, blueberry grow pot were predicted to be neutral. There were five rapidly changing sites in the M domain and six sites undergoing positive selection in the I domain of FUL1. None of the sites in the M domain showed a change in polarity.

Only one site in the I domain showed a change in polarity, but this site was predicted to be neutral functionally. MBP10 proteins had 20 sites undergoing diversifying selection in the K domain, only 1 such site in the M domain and 3 in the I domain .Of these, only three sites in the I domain showed a change in charge, all of which were also predicted not to have a negative effect on function.We compared euFUL expression data for the cultivated and wild tomato species, potato and Nicotiana benthamiana to identify any patterns that might be the result of changes in the regulatory regions following the duplications of these genes. Not all data from online sources were comparable across species, as different studies included different organs and developmental stages in their analyses, limiting cross-species comparisons. The analysis shows similar spatial expression patterns for FUL1 and FUL2 . These two paralogs are broadly expressed in leaves, flowers and fruits of tomato, potato, and tobacco. Although the eFP browser data shows no expression for FUL1 and FUL2 in tomato leaves, our RT-PCR data and previous publications show expression of all four euFUL homologs in these organs. Both euFULI genes are expressed relatively weakly in the roots of tomato, potato, and tobacco . Although spatial domains of expression are similar for the euFULI genes, they differ in temporal expression over the course of fruit developmental stages in tomato. Although both FUL1 and FUL2 are expressed in the fruits of all species, in tomato FUL2 is highly expressed during the early stages of fruit development and then tapers off, whereas FUL1 expression increases with time . In comparison to the euFULI genes, the two euFULII paralogs show more striking differences in spatial expression at the organ level , and also between species. In all species for which expression is reported, MBP10, alone among the euFUL genes in Solanaceae, is not expressed in fruits, or is expressed at barely detectable levels. In tomato, MBP20 is expressed strongly in roots while MBP10 is not. By contrast, in potato tubers, MBP10 expression is high and MBP20 is not expressed . The online sources and our RT-PCR data also show subtle intra-specific differences in expression between MBP10 and MBP20 in flowers . In addition, our RT-PCR data show that MBP10 is expressed relatively weakly in petals and stamens in tomato while MBP20 is expressed throughout the flower . However, these differences seem to be a matter of expression intensity in comparison to the more striking contrasts seen in roots, tubers, and fruits. The types of differences in expression between FUL1 and FUL2 versus MBP10 and MBP20 might be due to differences in the regulatory environment as a result of the different waysin which these duplicates arose. A tandem duplication and inversion may have disrupted regulatory regions in ways that would not be associated with a whole genome duplication or triplication . To investigate this, we searched for putative TF binding sites in the promoter regions of euFUL genes in tomato, potato, and woodland tobacco to compare the differences between the pairs of paralogs . Woodland tobacco was used rather than N. benthamiana since relatively longer promoter sequence lengths for euFUL genes were available for this genome assembly . Despite this, the maximum available promoter length for NsMBP10 was about 3.3 kb. We found that the differences in types and numbers of predicted TF binding sites between FUL1 and FUL2 were comparable to the differences between MBP10 and MBP20 . Nonetheless we did find some differences that may underlie observed differences in expression between paralogs. Some of these differences were presence/absence of binding sites for a particular TF, and some were in the number and distribution of sites. Putative binding sites for AUXIN RESPONSE FACTORS were absent from the tomato FUL2 promoter while they were present in the promoters of all other euFUL genes in all species examined. Only FUL2 in tomato, FUL1 in potato, and MBP10 in woodland tobacco contained binding sites for STOREKEEPER .

ETHYLENE INSENSITIVE 3 has three sites in tomato FUL1 and five in tomato FUL2, but the distribution of the sites differs. In FUL1, there are no sites within 2 kb of the coding sequence, and three within 5 kb, whereas in FUL2 there is one site in the 2 kb region and four in the full 5 kb region. In woodland tobacco, there are three EIN3 sites in FUL1, all of which are within the 2 kb region, and only one in FUL2, which is located between 2 and 5 kb. These types of differences may underlie observed differences in expression.In Solanaceae, there has been a major shift to fleshy fruit in the Solanoideae . However, we do not know the molecular basis of this economically and ecologically important evolutionary event. FUL negatively regulates lignification in the dehiscence zone in the dry silique of A. thaliana, and functions in cauline leaf development, the transition to flowering and determinacy . Studies of FUL ortholog function across the angiosperms have shown that it is labile, and orthologs have acquired diverse roles over evolutionary time. VEGETATIVE 1 , an ortholog of FUL in pea , is involved in secondary inflorescence meristem identity . AGAMOUS-like 79 , the A. thaliana euFULII paralog of FUL, is mainly expressed in the root and has functions in lateral root development and may also play a role function in leaf shape, leaf number, branching, and time to flowering . However, the over expression of an AGL79 ortholog from snapdragon in A. thaliana resulted in indehiscent siliques, suggesting a role more similar to A. thaliana FUL . Evidence suggests that in tomato, one of the AGL79 orthologs, MBP20, plays a role in leaf development . VERNALIZATION 1 genes, which are FUL-like orthologs in grass species such as wheat and barley , function in the vernalization response . Evidence to date, therefore, suggests that euFUL function is labile, and has changed substantially in different plant lineages during the course of angiosperm evolution. Thus it is not surprising to find a change in function of euFUL orthologs in Solanaceae. There is evidence to suggest that Solanaceae euFUL orthologs play a role similar to that of A. thaliana FUL in the development of dry dehiscent fruits . However, studies suggest that in the fleshy fruit of Solanoideae, FUL orthologs play roles in pigmentation as well as ethylene response, cell wall modification, glutamic acid degradation, volatile production, and pericarp and cuticle thickness .

It is possible that these negative regulators induce parthenocarpy as in the down-regulation of SlARF7 ovary transcript after pollination in tomato .A gene encoding an AOS in the jasmonate biosynthesis pathway was highly down-regulated in seedless transgenic fruits. Since published data associate jasmonates with early stages in climacteric fruit ripening and triggering ethylene production , it is of interest to determine whether seedless transgenic fruits differ in the rate of fruit ripening or in shelf life. Among genes with differential expression in transgenic fruit, some highly down-regulated genes may have important functions in fruit development. Fifteen down-regulated genes were found in parthenocarpic transgenic fruits that were involved in cell wall metabolism . Two of these, GDP-mannose pyrophorylase and b-galactosidase, were highly down-regulated, and a b-1,3 glucan hydrolase was significantly up-regulated in seedless fruits. The effect of these expression changes merits further investigation, since in tomato many genes may cause fruit softening . Indeed, additional cell wall hydrolases and expansins have been associated with tomato fruit softening . Another important functional category among differentially expressed genes was minor CHO metabolism involved in fruit sugar partitioning. The metabolomic analysis found no differences in sugars, but this was not verified in ripe fruit. Many proteins in these pathways are allosterically regulated, so their activity in the fruit may be less affected by changes in transcript level. To validate these data with TaqMan real-time PCR analysis, 17 genes were analysed for correspondence between microarrays and real-time PCR . Twelve of the 17 genes showed a microarray versus real-time RT-PCR correlation of >0.75 and the remaining five genes showed a lower correlation.

These five genes were the high affinity calcium antiporter CAX1 , sugar transporter , L-lactate dehydrogenase , dutch bucket hydroponic shortchain dehydrogenase reductase , and putative vicilin . However, genes involved in auxin and ethylene biosynthesis and signalling were con- firmed to be differentially regulated between transgenic and wild-type fruits.The next sep was to address how the changes in gene expression altered the overall metabolomic profile of parthenocarpic transgenic fruit. The concentrations of >400 metabolites in parthenocarpic fruits transformed with the four different constructs were compared with those from 12 wild-type fruits containing seeds. The acquired data sets were compared using PCA to determine differences and similarities among transgenic seedless fruits and seeded wild-type fruits at the breaker stage. Linear combination of metabolic data generated new vectors or groups to best explain overall variance in the data set without prior assumptions about whether and how clusters might form. It was immediately clear that the overall metabolomic data did not show clear differences among the different fruit genotypes . PCA could not separate the four transgenic lines and the controls: the 95% confidence intervals of the four treatments and two controls overlapped. Principal component 1, which accounted for ;38% of the variance, partly distinguished the control from some parthenocarpic lines such that all negative values were from transgenic lines, but the separation was not complete. Principal component 2 did not clearly separate any treatments from the controls. The relative concentrations of >400 metabolites were determined by peak area in transgenic and control fruits. However, many of them do not have a completely determined structure and could not be identified as a known molecule. Metabolites with known structure were divided into important functional categories and they were compared with transgenic seedless and wild-type seeded fruits using ANOVA univariate analysis .

It was expected that most differences at a metabolomic level induced by the transgene expression might occur at the beginning of fruit set and before fruits reached their final size. However, some changes are also expected when fruitsreach the breaker stage. This stage is physiologically very active, crucial for the ripening process, and important for the development of fruit quality phenotypes. Among 400 compounds analysed, only 16 showed significant differences between transgenic and wild-type fruits . Three of 19 amino acids showed significant differences . INO-rolB-transformed fruits had higher concentrations of these three amino acids than other seedless and seeded fruits. Six of the 18 acids determined revealed significant differences among different fruits. INO-rolB fruits had significantly more glutamate, malate, fumarate, and ascorbate than the other transgenic and seeded wild-type fruits. Among fatty acids, DefH9- iaaM and rolB-transformed fruits had significantly more stearic acid and palmitic acid than seeded wild-type fruits. Linoleic acid was also significantly higher in all transgenic fruits than in seeded wild-type fruits. Among other metabolites, rolB fruits had more oxoproline and ethanolamine and INO-rolB fruits had more putrescine than seeded wild-type fruits. There were no significant differences in sugars among transgenic and wild-type fruits. Despite the differences in gene expression among transgenic and control fruit, PCA analysis of 400 metabolites showed that the overall metabolomic analysis did not distinguish transgenic fruit from untransformed controls . Analysis was performed in fruits at a breaker stage and it would be interesting to determine what occurs also in the ripe fruits. Since only 16 metabolites showed significant differences between transgenic and wild-type fruits, the fundamental metabolism of the fruit seemed to be mostly unchanged. However, some important metabolites were higher in parthenocarpic than in wild-type fruits, especially in INO-rolB fruits, which had the most variability among biological replicates. Although all fruits were harvested at the breaker stage, such biological variability was expected due to unavoidable small differences in fruit developmental stage.

However, it is also possible that these metabolite differences were due to ovule driven expression of rolB regulating rolB-specific fruit metabolic pathways. Fatty acids were significantly higher in DefH9-iaaM and rolB-transformed fruits than in seeded wild-type fruits. Linoleic acid was also significantly higher in all transgenic fruits than in seeded wild-type ones. These data are coincident with differences observed in transcripts related to lipid metabolism. In Arabidopsis, auxins and cytokinins induce FAD3, a desaturase gene that alters fatty acid composition . Several genes involved in auxin metabolism were differentially regulated in our transgenic seedless fruits than in wild-type fruits: some were down-regulated and some up-regulated. Although Yamamoto reported that fatty acid desaturases are auxin regulated in mung bean, auxin regulation of fatty acid biosynthesis is not fully understood.Cracking of the epidermis of harvested fruit destroys the appearance and increases the susceptibility of fruit to infections by opportunistic pathogens. Fruit with cracks are not marketable, and, therefore, have reduced economic value. Fissures of the fruit epidermis often occur prior to harvest, but can also occur after harvest, depending on storage and environmental conditions. The predisposition to form cracks has been correlated with heredity, various fruit traits and external causes, such as cultivation practices and growing environment . Many researchers have attributed cracking predisposition to the thickness of the fruit’s cuticular layer adjacent to the epidermal and sub-epidermal cells. Cracking has also been linked to the loss of flesh firmness and cell wall integrity. Fruit that are susceptible to cracking often have high levels of soluble solids and produce juice with elevated concentrations of osmotically active compounds. As fruit ripen, there is a dramatic increase in their tendency to crack. The production of large, uncracked, ripe fruit in cultivars with thin skins and high soluble solids has proven to be an unmet challenge. The complexity and structural plasticity of the ripening process are challenges for approaches designed to understand the relationship between ripening-associated softening, dutch buckets system sugar accumulation and cracking. Considerable reductions in the incidence and degree of fruit cracking may be achieved by changing cultural or post harvest practices. Consistent watering or exogenous applications of boron, calcium and/or growth promoters, such as GA3, can reduce cracking. Applications of calcium and boron strengthen the linkages between polysaccharides in the cell wall, increasing firmness. Applications of GA3 likely decrease cracking because this treatment increases the deposition of cuticular material in the epidermis and makes it more elastic. Treating plants with abscisic acid increases water movement into and promotes enlargement of the fruit. ABA treatment also increases the tendency of fruit to crack. Application of ABA to “Cabernet Sauvignon” grape berries promotes ripening and the expression of PG1 and proline-rich cell wall protein genes, typically expressed during ripening. Cracking in tomato fruit most commonly begins as they ripen.

During ripening, cell wall modifying proteins, including polygalacturonases and expansins , cooperatively disassemble wall polysaccharide networks and, thereby, contribute to the softening of fruit. Differences in cell wall structure between varieties and between unripe and ripe fruit could be an important factor in fruit tendency to crack. Quantitative and qualitative changes in the sugars in ripe fruit could influence water potential and also contribute to the tendency of the fruit to crack. Over expression of a Golden 2-like transcription factor, SlGLK2, in tomato enhances chloroplast elaboration and photosynthesis gene expression in developing fruit, and results in ripe fruit with elevated soluble solids content. It is desirable to breed or select for varieties whose fruit resist cracking under diverse environmental conditions without hormone treatments. Therefore, we investigated whether reducing the simultaneous expression of SlPG and SlEXP1 genes affects the tendency of fruit to crack. We were also interested to observe cracking of tomato lines with functional or non-functional forms of SlGLK2 to explore the contributions of solutes and sugars to the fruit’s predisposition to form cracks. ABA was used as a tool to enhance cracking incidence of the tomato fruit.A preliminary experiment was conducted in 2012, followed by a similar but more extensive experiment in 2013 with 3 genotypes. The Alisa Craig S. lycopersicum cultivar  expresses functional SlPG, SlEXP1 and SlGLK2 genes. The transgenic line, pg/exp, has suppressed ripe fruit expression of both SlPG and SlEXP1. Suppression of SlPG or SlEXP1 alone did not significantly enhance fruit firmness. However, fruits with suppressed expression of both genes were significantly firmer throughout ripening with a long-term storage and more viscous juice than control fruits. The monogenic u/u mutant of AC, “Craigella” , contains a mutation in SlGLK2 that results in a truncated and, therefore, nonfunctional glk2 protein. In the 2012 experiment, plants of the pg/exp and glk2 genotypes were grown from 15 December 2011 to 3 May 2012 in greenhouses at the University of California, Davis. Prior to germination, seeds were soaked for 3 h in water and for 30 min in a 10% solution of bleach to reduce potential viral contamination, then washed 3 times with deionized water and placed into Petri dishes with 7 mL 30 µM GA3 for 2 days at 4 °C. Subsequently, seeds were germinated in a growth chamber at 25 °C. Seedlings were transplanted and moved to the greenhouse on 16 January. There were 64 plants of each genotype subdivided into two treatments and 4 replications with 8 single plant replicates per treatment. Seedlings were grown in 9.5-L pots containing 33.3% each peat, sand, and red wood compost with 2.6 kg dolomite lime m−3 . The plants were irrigated twice per day with 350 mL of UC Davis nutrient solution containing NH4 +, NO3 − , H2PO4 − , K+ , Ca2+ , Mg2+ , SO4 2− , Fe , Mn , B , Cu , Zn and Mo . Plants were pollinated on 8 March 2012, and were topped on 15 March 2012 when they had 2 clusters of flowers. On 18 April, ABA and control spray treatments began. The plants were sprayed 1× per week for 3 weeks with a backpack applicator until the plants were completely covered with a solution containing deionized water or 0.5 mg L−1 ABA ; each solution also contained 0.5 mL L−1 polysorbate 20 as a surfactant. The cracking fruits were counted and cracking rates were calculated on 26 April. The other characteristics of the fruits were then analyzed. In 2013, WT, pg/exp and glk2 plants were grown from 17 December 2012 to 6 May 2013 in greenhouses. Seed germination protocols were like those used in 2012. Seedlings were transplanted into pots in the greenhouse on 14 January. There were 192 plants in total with 64 plants for each genotype, as in 2012. In the greenhouse, passive ventilation was used to maintain a relative humidity of 26.1–27.4%. The average temperature ranged from 21.5 to 22.7 °C with minimum of 12.8 °C and maximum of 35.0 °C. Cultivation practices were the same as in 2012, although the irrigation schedule was modified due to growth periods. Plants were initially irrigated twice per day with 350 mL of UC Davis nutrient solution.

Pulp cells also have a much higher set of transcripts involved in carbohydrate metabolism, but a lower set of transcripts involved in lipid, amino acid, vitamin, nitrogen and sulfur metabolism than in the skins. Hormones can influence berry development and ripening. Concentrations of auxin, cytokinins and gibberellins tend to increase in early fruit development of the first stage. At veraison, these hormone concentrations have declined concomitant with a peak in abscisic acid concentration just before veraison. Auxin prolongs the Stage 2 lag phase and inhibits anthocyanin biosynthesis and color development in Stage 3. Grapevine, a non-climacteric fruit, is not very sensitive to ethylene; however, ethylene appears to be necessary for normal fruit ripening. Ethylene concentration is highest at anthesis, but declines to low levels upon fruit set; ethylene concentrations rise slightly thereafter and peak just before veraison then decline to low levels by maturity. Ethylene also plays a role in the ripening of another non-climacteric fruit, strawberry. ABA also appears to be important in grape berry ripening during veraison when ABA concentrations increase resulting in increased expression of anthocyanin biosynthetic genes and anthocyanin accumulation in the skin. ABA induces ABF2, a transcription factor that affects berry ripening by stimulating berry softening and phenylpropanoid accumulation. In addition, ABA affects sugar accumulation in ripening berries by stimulating acid invertase activity and the induction of sugar transporters. It is not clear whether ABA directly affects flavor volatiles , strawberry gutter system but there could be indirect effects due to competition for common precursors in the carotenoid pathway.

Many grape berry ripening studies have focused on targeted sampling over a broad range of berry development stages, but generally with an emphasis around veraison, when berry ripening is considered to begin. In this study, a narrower focus is taken on the late ripening stages where many berry flavors are known to develop in the skin. We show that that the abundance of transcripts involved in ethylene signaling is increased along with those associated with terpenoid and fatty acid metabolism, particularly in the skin.Cabernet Sauvignon clusters were harvested in 2008 from a commercial vineyard in Paso Robles, California at various times after veraison with a focus on targeting °Brix levels near maturity. Dates and metabolic details that establish the developmental state of the berries at each harvest are presented in Additional file 1. Berries advanced by harvest date with the typical developmental changes for Cabernet Sauvignon: decreases in titratable acidity and 2- isobutyl-3-methoxypyrazine concentrations and increases in sugar and color . Transcriptomic analysis focused on four harvest dates having average cluster °Brix levels of 22.6, 23.2, 25.0 and 36.7. Wines made in an earlier study from grapes harvested at comparable levels of sugars or total soluble solids to those in the present study showed clear sensory differences. Six biological replicates, comprising two clusters each, were separated into skins and pulp in preparation for RNA extraction and transcriptomic analysis using the NimbleGen Grape Whole-Genome Microarray. Thus, a 4 × 2 factorial experimental design was established. After standard microarray processing and data normalization, two-way ANOVA indicated that the transcript abundance of 16,280 transcripts statistically significantly changed across the °Brix levels below the adjusted p-value of 0.05 , the transcript abundance of 10,581 transcripts changed significantly across Tissue types, and the abundance of 2053 transcripts changed significantly with respect to the °Brix x Tissue interaction term p-value column: adjBrix, adjTissue or adjTissue*Brix.

A note of caution must be added here. There are high similarities amongst members in certain Vitis gene families , making it very likely that cross-hybridization can occur with probes on the microarray with high similarity to other genes. We estimate approximately 13,000 genes have the potential for cross-hybridization, with at least one probe of a set of four unique probes for that gene on the microarray potentially cross-hybridizing with probes for another gene on the microarray. Genes with the potential for cross hybridization have been identified and are highlighted in light red in Additional file 2. The rationale to include them is that although individual genes can not be uniquely separated, the probe sets can identify a gene and its highly similar gene family members, thus, providing some useful information about the biological responses of the plant. An additional approach was taken, removing cross-hybridizing probes before quantitative data analysis . Many of the significant genes were unaffected by this processing, but 3600 genes were completely removed from the analysis. Thus, it was felt that valuable information was lost using such a stringent approach. The less stringent approach allowing for analysis of genes with potential cross hybridization was used here in the rest of the analyses. To assess the main processes affected by these treatments, the gene ontologies of significantly affected transcripts were analyzed for statistical significance using BinGO. Based on transcripts that had significant changes in abundance with °Brix level, 230 biological processes were significantly over represented in this group . The three top over represented processes were response to abiotic stress, bio-synthetic process, and response to chemical stimulus, a rather generic set of categories. Tissue differences were more revealing at the stage when flavors peak; 4865 transcripts that were significantly higher in skins compared to pulp at 23.2 °Brix were tested for over represented GO functional categories .

Some of the top GO categories included photosynthesis, isoprenoid biosynthesis, and pigment biosynthesis . Some of the transcripts with the largest differences between skin and pulp at 23.2 °Brix are β-ketoacyl-CoA synthase , taxane 10-β-hydroxylase , wax synthase, a lipase, an ABC transporter, and phenylalanine ammonia-lyase . The abundance of 5716 transcripts was significantly higher in pulp than skin at 23.2 °Brix . Some of the top GO categories over represented were a variety of transport processes and small GTPase mediated signal transduction . Some of the transcripts with the largest differences in abundance with pulp greater than skin at 23.2 °Brix were polygalacturonase , flavonol synthase, stachyose synthase, an amino acid transporter, a potassium channel , and HRE2 . The transcript abundance of 2053 genes had significantly differential expression across °Brix levels and tissues . The top GO categories over represented in this set involved photosynthesis and phenylpropanoid metabolism, both associated with the berry skin . Other flavorcentric categories of the 57 categories over represented include aromatic compound biosynthesis, fatty acid metabolism and alcohol catabolism. This transcript set was further analyzed by dividing into 10 clusters using k-means clustering . The over represented GO categories were determined for each cluster . Eight of the 10 clusters had distinct over represented GO categories; two clusters did not have any over represented GO categories, meaning that the genes in these two clusters were assigned to GO categories of expected proportions when compared to the entire NimbleGen array. Clusters 1, 8, 9 and 10 had a large number of over represented categories. Many GO categories within a cluster are subsets of others in that cluster and were grouped together. For example, cluster 4 had four over represented GO categories, oxygen transport, gas transport, heat acclimation and response to heat. The four categories could be grouped into two, as two are subsets of the others; this is how they were listed in Table 1. As we were interested in compounds associated with berry flavors as they develop or change in the late stages of berry ripening, we took a more targeted approach for analysis with this in mind. Berries at 24° Brix are known to be near-optimal for flavor, grow strawberry in containers thus we took a simple approach to look for genes that were peaking around this stage. We found some significant and large increases in transcript abundance between the 22.6 and 23.2 °Brix levels. A group of VviERF6 transcription factor paralogs represented 6of the top 10 transcripts increasing in transcript abundance from 22.6 to 23.2 °Brix in the skin, but not in the pulp . These VviERF6 TFs were also found in Cluster 8 . This is very interesting since many flavor compounds are derived from the skin and ERF TFs are known to be responsive to ethylene, a known fruit-ripening hormone. These VviERF TFs were named ERF105 in the annotation by Grimplet et al. , however they are more orthologous with AtERF6 as determined by a more comprehensive phylogenetic method using many plant species at Gramene . Annotation details of the V1 gene models of the VviAP2/ERF superfamily can be found in Additional file 8 including updated Vvi symbols according to its closest Arabidopsis ortholog as instructed by the Grapevine Gene Nomenclature System developed by the International Grape Genome Program Supernomenclature committee.

This renaming of the AP2/ERF superfamily should facilitate comparative analyses and functions with other species, particularly Arabidopsis.This study focused on the very late stages of the mature Cabernet Sauvignon berry when fruit flavors are known to develop. Cabernet Sauvignon is an important red wine cultivar, originating from the Bordeaux region of France. It is now grown in many countries. Wines made from Cabernet Sauvignon are dark red with flavors of dark fruit and berries. They also can contain herbaceous characters such as green bell pepper flavor that are particulary prevalent in underripe grapes. Grape flavor is complex consisting not only of many different fruit descriptors, but descriptors that are frequently made up of a complex mixture of aromatic compounds. For example, black currant flavor, in part, can be attributed to 1,8-cineole, 3-methyl-1-butanol, ethyl hexanoate, 2- methoxy-3-isopropylpyrazine, linalool, 4-terpineol, and β- damascenone and major components of raspberry flavor can be attributed to α- and β-ionone, α- and β- phellandrene, linalool, β-damascenone, geraniol, nerol and raspberry ketone. Some common volatile compounds found in the aroma profiles of these dark fruits and berries include benzaldehyde, 1-hexanol, 2-heptanol, hexyl acetate, β-ionone, β-damascenone, linalool, and α-pinene. In a study of Cabernet Sauvignon grapes and wines in Australia, Cabernet Sauvignon berry aromas were associated with trans-geraniol and 2-pentyl furan and Cabernet Sauvignon flavor was associated with 3-hexenol, 2-heptanol, heptadienol and octanal. In another comprehensive study of 350 volatiles of Cabernet Sauvignon wines from all over Australia, the factors influencing sensory attributes were found to be complex; in part, norisoprenoids and δ − and γ-lactones were associated with sweet and fruity characteristics and red berry and dried fruit aromas were correlated with ethyl and acetate esters. In Cabernet Sauvignon wines from the USA, sensory attributes were complex also and significantly affected by alcohol level of the wine. Linalool and hexyl acetate were postitively associated with berry aroma and IBMP was positively correlated with green bell pepper aroma. In France, β-damascenone was found to contribute to Cabernet Sauvignon wine aroma. Thus, flavor development in berries and wines is very complex, being affected by a large number of factors including genetics, chemistry, time and environment. In this paper we begin to examine the changes in transcript abundance that may contribute to flavor development. We show that the transcript abundance of many genes involved in fatty acid, carotenoid, isoprenoid and terpenoid metabolism was increased in the skin and peaked at the °Brix levels known to have the highest fruit flavors . Many of these are involved in the production of dark fruit flavors such as linalool synthases, carotenoid dioxygenases and lipoxygenases. These genes serve as good candidates for berry development and flavor markers during ripening. A broader range of studies from different cultivars, locations and environments are needed to determine a common set of genes involved in berry and flavor development. A similar study was conducted on the production of volatile aromas in Cabernet Sauvignon berries across many developmental stages, including a detailed analysis of the °Brix levels that was surveyed in this study. They found that the production of alcohol volatiles from the lipoxygenase pathway dominated in the later stages of berry ripening and suggested that the activity of alcohol dehydrogenases also could play an important role. The abundance of the transcript of VviOMT1 decreased in the pulp with increasing °Brix level and was correlated with IBMP concentrations in the late stages of berry development in this study. Both OMT1 and OMT3 have been shown to synthesize IBMP. Furthermore, the transcript abundance of each gene has been correlated with IBMP concentration, but the transcript abundance of each gene cannot fully account for the total IBMP present in all genotypes and conditions.

Ohmic heating. Ohmic heating is a thermal processing method in which an alternating electrical current is passed through food products to generate heat internally. Ohmic heating is said to produce a uniform, inside-out heating pattern that heats foods faster and more evenly than conventional outside-in heating methods. According to Lima et al. , potential applications for ohmic heating relevant to fruit and vegetable processing include blanching, evaporation, dehydration, fermentation, and extraction. In tests at the Louisiana State University Agricultural Center, sweet potato samples were processed using ohmic heating prior to freeze drying. Ohmic heating reportedly increased the rate of freeze-drying up to 25% compared to samples that did not undergo ohmic heating, which led to significant savings in both processing time and energy use . However, ohmic heating parameters such as the frequency of the alternating current, the applied voltage, the temperature to which the sample is heated, and the electrical conductivity of the food can all have a significant effect on the performance of the process. Infrared drying. In conventional drying methods, substantial amounts of air must be heated and circulated around the product to be dried. In contrast, infrared drying uses infrared radiation to heat only the material that needs to be heated—not the surrounding air—and thus saves energy compared to conventional methods. For drying apple slices, a comparison of infrared drying with convective drying done using equivalent processing parameters showed that energy costs were lower and that the time of the drying process could be shortened by up to 50% using infrared methods . Pulsed fluid-bed drying. The pulsed fluid-bed dryer is a modification of the conventional fluid-bed dryer . In pulsed fluid-bed drying, grow bucket gas pulses cause high-frequency vibrations within the bed of product particles.

Reported advantages of the pulsed fluid-bed drying approach include easier fluidization of irregular particle shapes, fluidization with 30% to 50% less air than conventional methods , and reduced channeling of particles . Additionally, pulsed fluid-bed dryers are roughly half the size of conventional conveyor-type dryers. Successful trial applications in the food industry include the drying of carrot cubes and the drying of chopped onions. In the drying of carrot cubes, a pulsed fluid-bed dryer reduced the total drying time by two to three times compared to traditional fluid-bed drying methods while providing a final product that was highly uniform in color and moisture content. Similarly, for chopped onions, the final products were of high color and reconstitution quality and uniform in moisture content . Pulsed electric field pasteurization. Pulsed electric field pasteurization for juices may provide superior taste and freshness compared to juices undergoing conventional heat treatment. In the pulsed electric field process, liquids are exposed to high voltage pulses of electricity to inactivate harmful micro-organisms as well as enzymes that degrade the quality of fruit juices over time. The energy savings associated with pulsed electric field processing arise from the fact that the process operates at lower temperatures than conventional heat-based pasteurization methods and thus the pasteurized juices require less cooling energy . Pulsed electric field pasteurizing has been successfully employed by the Genesis Juice Corporation of Eugene, Oregon, in the production of organic bottled fruit juices . The company reported that the major motivation for using the new technology was to avoid the loss of flavor associated with conventional thermal pasteurization methods.Geothermal heat pumps for HVAC. 

Geothermal heat pumps take advantage of the cool, constant temperature of the earth to provide heating and cooling to a building. To date, most applications of geothermal heat pumps have been in the residential and commercial sectors rather than in the industrial sector. However, geothermal heat pumps may be a viable replacement for traditional HVAC systems in office or warehouse spaces in the fruit and vegetable processing industry. In winter, a water solution is circulated through pipes buried in the ground, which absorbs heat from the earth and carries it into the building structure. A heat pump system inside the building transfers this heat to air that is circulated through the building’s duct work to warm the interior space. In the summer, the process is reversed: heat is extracted from the air in the building and transferred through the heat pump to the underground piping, where heat is transferred back to the earth. The only external energy needed is a small amount of electricity to operate fans and ground loop pumps . The Geothermal Heat Pump Consortium claims that the technology can reduce space heating and cooling energy consumption by 25% to 50% compared to traditional building HVAC systems.Carbon dioxide as a refrigerant. In the food industry, CO2 can be used for quick freezing, surface freezing, chilling, and refrigeration. In cryogenic tunnels and spiral freezers, high pressure liquid CO2 is injected through nozzles that convert it to a mixture of CO2 gas and dry ice that covers the surface of the food product. Liquid CO2 is reported to generate faster cooling rates than conventional freezing processes. In addition, liquid CO2 freezing equipment eliminates the need for compressor systems, thereby taking up less room than comparable mechanical freezers. Since 2001, the frozen vegetable producer Ardo B.V., located in Zundert, the Netherlands, has been operating a 560 kW combined ammonia-CO2 freezer, which uses ammonia in the higher temperature range and CO2 in the lower temperature range.

The energy savings of this system, in comparison to a conventional ammonia-based expansion system, have been estimated at around $66,000 per year. The estimated payback period is 11 years. .In many U.S. fruit and vegetable processing facilities, water is a resource that can be just as critical and costly as energy in the production process. Water is used throughout the fruit and vegetable processing industry for process cooling, boiler systems, water fluming, blanching, peeling, cooking, product rinsing, and equipment cleaning, as well as in the products themselves as a primary ingredient . In California alone, the water consumption of the fruit and vegetable processing industry has been estimated at nearly 23 billion gallons per year . The specific water usage required in fruit and vegetable processing depends heavily on the type of product manufactured as well as on the water management practices at individual facilities. Reported values of specific water usage in the U.S. fruit and vegetable processing industry range from several hundred gallons per ton of product to tens of thousands of gallons per ton of product . This range suggests significant variation in water usage across the industry. According to a study by the World Bank , however, good facility water management programs can often help reduce specific water usage to the “best practice” levels indicated in Table 15.1 for different processed fruit and vegetable products.This chapter provides a brief overview of basic, proven water efficiency measures applicable to typical fruit and vegetable processing plants. In addition to reducing facility utility bills for water purchases, improved water efficiency can also lead to reduced energy consumption for water pumping and treatment, reduced wastewater discharge volumes, dutch bucket for tomatoes and reduced wastewater treatment costs. Furthermore, the recovery and recycling of water can also provide opportunities for energy recovery, which can help to further reduce facility energy costs. Water efficiency also reduces loads on local fresh water and wastewater treatment plants, which leads to indirect energy savings in the industrial water supply chain. According to Envirowise, a UK government program that promotes business resource efficiency, fruit and vegetable processing companies that have not implemented any water saving measures can often reduce water and effluent costs by 50% through water efficiency programs . Companies that have already implemented some measures—but not a systematic approach—can often still achieve a 20% decrease in water and effluent costs.Use of water efficient building fixtures. For building fixtures such as toilets, showers, and faucets, water efficient designs can be installed that lead to significant water savings. For example, low-flow toilets typically require only 1.6 gallons per flush, compared to 3.5 gallons per flush required for standard toilets . Additional options include low-flow shower heads, aerating faucets, self-closing faucets, and proximity sensing faucets that turn on and off automatically.24 Dry conveyors. Where feasible, water flumes might be replaced by belt conveyors or chutes to save significant quantities of water . However, the applicability of this measure will depend on the extent to which existing water flumes are integrated with other facility processes , how susceptible the product is to bruising or damage, and the flexibility of the installed equipment layout. Use of small diameter hoses. All applications of hoses should be assessed, and, where feasible, the smallest possible diameter hoses should be installed. Small diameter hoses provide a low flow, high pressure condition, which can reduce the volume of water required for a given task .

Air cooling. The use of air cooling instead of water cooling can lead to water savings in situations where air is a feasible process cooling alternative . However, from an energy perspective, water cooling is generally preferable to air cooling . Thus, the switch to air cooling should be carefully examined for each prospective process application to determine whether or not a favorable compromise between energy use and water use exists. Use of automated start/stop controls. For end uses of water with intermittent demand, sensors can be employed to detect the presence of materials and to supply water only when it is required by the process. Such sensors will turn off water supplies automatically when not required and also during non-production periods, thereby saving water . Reducing demand for steam and hot water. Reducing the demand for steam and hot water not only saves energy but also reduces the need for treated boiler water. Typically, fresh water must be treated to remove contaminants that might accumulate in the boiler, so reducing demand not only decreases boiler water use, but can also reduce the amount of purchased chemicals for boiler water treatment . The combined energy, water, and chemicals savings associated with reducing steam and hot water demand make it a particularly attractive measure. Steam and hot water demand can be reduced through the general steam system energy efficiency strategies discussed in Chapter 7 of this Energy Guide, as well as through process specific modifications. For example, where feasible, dry caustic peeling methods can be employed in lieu of wet caustic peeling or steam-based peeling methods to reduce process water consumption. Dry caustic peeling has been shown to reduce water consumption by up to 75% compared to wet caustic peeling in the processing of beets . Additional examples include the use of air cooling instead of water cooling to cool products after blanching, or the use of steam-based blanching methods instead of water-based blanching methods. Reducing cooling tower bleed-off. Cooling tower “bleed-off” refers to water that is periodically drained from the cooling tower basin to prevent the accumulation of solids. Bleed-off volumes can often be reduced by allowing higher concentrations of suspended and dissolved solids in the circulating water, which saves water. The challenge is to find the optimal balance between bleed-off and makeup water concentrations without forming scales. The water savings associated with this measure can be as high as 20% . The Ventura Coastal Plant, a manufacturer of citrus oils and frozen citrus juice concentrates in Ventura County, California, was able to increase the concentration ratios of its cooling towers and evaporative coolers such that bleed-off water volumes were reduced by 50%. The water savings amounted to almost 5,200 gallons per day, saving the company $6,940 per year in water costs . With capital costs of $5,000, the simple payback period was estimated at around seven months.Dry cleaning of equipment and surfaces. Fruit and vegetable wastes and residues should be removed manually from floors and equipment before the application of cleaning water to reduce water consumption. Dry cleaning can be done using brushes, squeegees, brooms, shovels, and vacuums. Often, solid and liquid wastes are chased down floor drains using a hose; a better practice is to use brooms or shovels and to dump wastes into a container designated for solid waste . High pressure low volume sprays. In applications such as truck, container, surface, and floor cleaning, total water consumption can be reduced by using high pressure low volume spray systems, which employ small diameter hoses and/or flow restricting spray nozzles. Such systems can also be fitted with manual triggers, which allow personnel to regulate use, or automatic shut-off valves to further reduce water consumption .

The company installed controls consisting of sensors and computer software, which automatically modulated compressor discharge and suction pressures to improve the coefficient of performance and to better adjust compressor operation to changes in refrigeration system cooling demand. The upgrade led to annual energy savings of 367,000 kWh as well as reduced operations and maintenance costs through more efficient system operation . The reported payback period, which included both electricity bill savings and reduced operations and maintenance costs, was around 2.6 years. Floating head pressure control. Floating head pressure control can be a particularly effective control strategy for reducing compressor energy consumption. Floating head pressure control allows compressor head pressures to move up or down with variations in ambient wet-bulb temperature, saving energy compared to fixed head pressure operation. However, additional energy is required for the condenser fan, which must be balanced with compressor energy savings. It is also important not to allow head pressure to go too low, as certain system demands might require minimum head pressures . Hackett et al. estimate a typical payback period of less than one year for floating head pressure control systems. A U.S. DOE sponsored energy audit at the Odwalla Juice Company’s facility in Dinuva, California, estimated that the use of floating head pressure control on the facility’s seven ammonia compressors would save the company nearly $108,000 per year in energy costs . Total estimated electricity savings were around 1 million kWh per year at a payback period of only six months. Birds Eye Walls, a UK based manufacturer of frozen foods, nft growing system implemented refrigeration controls that allowed for floating head pressure in its Gloucester, England, facility in 1994.

The controls led to a 30% lower head pressure on average, allowing the company to save around £150,000 in refrigeration costs annually . At an initial investment cost of less that £30,000 , the payback period was less than three months. Indirect lubricant cooling. Direct injection of refrigerant is an inefficient method for compressor cooling that can decrease the overall efficiency of screw-type compressors by as much as 5% to 10% . An indirect system is a more efficient option for lubricating and cooling screw-type compressors, in which a heat exchanger is used in conjunction with cooling tower water, a section of an evaporative condenser, or a thermosyphon system to cool compressor lubricant. Raising system suction pressure. In two-stage compressor systems, a simple way to save energy is to raise the suction pressure and temperature of the low-stage compressor when ambient temperatures decrease. It has been estimated that energy savings of about 8% can be realized in two-stage systems when suction temperatures are raised from -30 °F to -20 °F . Adjustable-speed drives on compressor motors. Adjustable-speed drives can be used in conjunction with control systems to better match compressor loads to system cooling requirements. The Industrial Refrigeration Consortium reports that ASDs used on compressors below a part-load ratio of about 95% will deliver performance equal to a fixed speed compressor but with lower electricity requirements. However, at near full load, ASDs are approximately 3% less efficient than fixed speed drives due to electrical power losses associated with the ASD controller. Adjustable-speed drives are thus most beneficial for refrigeration systems with large differences between required and installed condenser capacities . Galitsky et al. have estimated average refrigeration system energy savings of 10% from the use of ASDs on compressors. Naumes, Inc., an Oregon based company specializing in fruit growing, processing, storage, and juice production, recently upgraded their ammonia-based refrigeration system with computer controls and ASD compressors for more efficient matching of cooling demand and system load.

The new system saved the company a reported 741,000 kWh per year, with total annual energy savings of around $37,000 . The simple payback period was estimated at just over two years. As part of a planned expansion for its dairy facility in Portland, Oregon, WestFarm Foods installed a new compressor with a 350 hp ASD, which allowed the remaining system compressors to either be off or working efficiently at 100% load. Other upgrades included new refrigeration system controls and ASDs on the system’s evaporator fans. The totalsystem upgrade reduced annual refrigeration system energy consumption by nearly 40% and annual operating costs by around $75,000 . At an investment cost of $310,000, the payback period was estimated at roughly four years; however, energy efficiency investment incentives from Portland General Electric as well as a 35% tax credit from the Oregon Department of Energy helped reduce the final payback to around one year. In 2003, Oregon Freeze Dry, a manufacturer of freeze-dried fruits, vegetables, and other specialty foods, installed ASDs on its refrigeration system screw compressors at its Albany, Oregon, facility. The company also decided to replace an undersized eight inch suction line with a new 12 inch line. The energy savings of the ASD and suction line installations amounted to nearly 2 million kWh per year , while energy cost savings amounted to $77,700 per year . Compressor heat recovery. Where economically feasible, rejected heat can be recovered from compressors and used in other facility applications, such as space heating or water heating. Further details on this measure are provided in Chapter 10. Dedicating a compressor to defrosting. It has been reported that if one compressor of a large system can be dedicated to running at the pressure needed for the defrost cycle, while the other compressors can be run at lower system pressures, that the resulting energy savings can often justify the cost of the dedicated compressor .

Keeping condensers clean. Condensers should be checked regularly for dirt, ice buildup, or plugged nozzles, which can reduce heat transfer rates and thus raise the condensing temperature. Furthermore, water-cooled and evaporative condensers should be kept free of hard water or bacterial buildup, which can cause fouling, scaling, and clogging that can also lead to increased condensing temperatures. In general, a one degree Celsius increase in condensing temperature will increase operating costs by 2% to 4% . Badly corroded condensers should be replaced as soon as possible. Automatic purging of condensers. Periodic purging of evaporative condensers is needed to remove non-condensable gases , which can reduce refrigeration system efficiency by increasing system head pressure and impeding condenser heat transfer . Automatic purging systems can help refrigeration systems operate efficiently by ensuring purging occurs on a regular basis. Automatic purging systems can also reduce the refrigerant loss and labor costs associated with manual purging. Excel Logistics Ltd., an operator of cold storage facilities in the United Kingdom, installed a five-point automatic refrigeration purging system at their Glasgow, Scotland, facility in 1989. Previously, the company purged its system manually on a weekly basis, which was time consuming and often led to refrigerant loss. The automatic purging system featured computer controls and five different refrigeration system purge points: one at each end of thereceiver, one on each of the two condenser outlets, and one on the hot gas line. The company reported that the automatic purging system led to a 15% reduction in compressor energy use and £8,800 in annual energy savings . The simple payback period, nft hydroponic system including both energy and maintenance cost savings, was 10 months. Reducing condenser fan use. Sometimes condenser fans are operated continuously, even when the refrigeration system’s compressor isn’t running. This practice wastes energy. Wherever possible, the operation of condenser fans should be coupled to the operation of the system’s compressors to ensure that the fans are only run when needed. Reducing condensing pressure. This measure is similar to floating head pressure control for compressors . To reduce the energy required to compress refrigerant, condensing pressures and temperatures should be set as low as possible. Computer controls can be installed on condensing systems to minimize condensing temperatures and pressures based on ambient wet-bulb temperatures, as well as to optimize the use of condenser fans and water . Lowering the condensing temperature can reduce compressor energy use by around 2% to 3% for every degree Celsius of temperature reduction . Use of axial condenser fans. Air-cooled or evaporative condensers generally do not need high-pressure air, and thus axial fans are well suited for this application. Adjustable-speed drives on condenser fans. For refrigeration systems with large differences between installed and operating condensing capacity, the use of ASDs on condenser fans can lead to significant energy savings compared to fixed-speed condenser fans. Prior to installing ASDs, however, it is important to establish the extent to which the condensing pressure can be floated. On systems where floating head operation is stable, ASDs can lower condenser fan energy consumption by up to 40% compared to operating a fixed-speed condenser fan in on/off fashion .

Cycling of evaporator fans in cold storage. It is often possible to maintain adequate temperature in cold storage areas without continuously running evaporator fans. Where feasible, evaporator fans can be turned off or ramped down periodically using timers or variable-speed control systems to save electricity while still maintaining proper cold storage temperatures. The cycling of evaporator fans should be managed carefully, however, to avoid stratification and to ensure that solenoids are cycled properly . In 1996, Stahlbush Island Farms, a grower, canner, and freezer of fruits and vegetables in Corvalis, Oregon, installed timers to cycle the evaporator fans of its cold storage unit. Prior to the installation of the timers, evaporator fans were run close to 24 hours per day. By cycling the evaporator fans, the company was able to save around 133,000 kWh of electricity per year because the fans ran for fewer hours and the fan motors released less heat into the cold storage unit . The annual savings were estimated at $4,500 and, with a one-time implementation cost of $1,000, the simple payback period was around three months. Adjustable-speed drives on evaporator fans. Similar to ASDs on condenser fans, for refrigeration systems with excess evaporator capacity, the installation of ASDs can lead to significant energy savings compared to fixed-speed fans. The cost effectiveness of ASDs, however, depends on the number of hours the evaporator fans can be run under part-load conditions. In an analysis of a -20° Fahrenheit freezer with seven evaporators, the use of ASDs on evaporator fans at a load ratio of 50% required 20% lower power than fixed-speed fans under the same operating conditions . The U.S. DOE has supported the development of a simple evaporator fan controller for medium temperature walk-in refrigeration units, which is capable of varying fan speed is reported to reduce evaporator and compressor energy consumption by 30% to 50% . The controller regulates the speed of evaporator fan motors to better match cooling demands in the refrigeration cycle. The U.S. DOE estimates typical payback periods of one to two years. As of 2000, the controller had been installed in 300 refrigeration units and had led to cumulative energy savings of around $80,000. According to BC Hydro , evaporator fan controllers are not good candidates for freezers that run under 28° Fahrenheit, have compressors that run continuously, have evaporator fans that run on poly-phase power, and have evaporator fans of types other than shaded-pole and permanent-split-capacitor. Demand defrost. Evaporators should be defrosted only when necessary, as opposed to on timed schedules where defrosting occurs regardless of need. Defrosting cycles should ideally be based on coil pressure readings, where an increase in pressure drop indicates that frost is present on the coils and that defrosting is necessary . Water defrosting. Water defrosting is said to be more efficient than hot gas defrosting . In water defrosting, water is sprayed manually over the evaporator coils to remove frost. However, water defrosting must be managed properly to ensure that the water does not freeze on the evaporator coils.Compressed air generally represents one of the most inefficient uses of energy in U.S. industry due to poor system efficiency. Typically, the efficiency of a compressed air system—from compressed air generation to end use—is only around 10% . Because of this inefficiency, if compressed air is used, it should be of minimum quantity for the shortest possible time; it should also be constantly monitored and weighed against potential alternatives. Many opportunities to reduce energy consumption in compressed air systems are not prohibitively expensive; payback periods for some options can be extremely short. Energy savings from compressed air system improvements can range from 20% to 50% of total system electricity consumption .

Packaging processes are generally powered using a combination of electric motors, solenoids, and compressed air actuators.The typical processes employed in fruit and vegetable canning are depicted in Figure 3.1. For both fruits and vegetables, inspection, grading, and washing are generally the first processing steps. Vegetables are then typically peeled if needed, subjected to size reduction to obtain the proper form, and blanched to inactivate enzymes. Immediately after blanching, vegetables are typically cooled in a water bath to prevent overcooking. For some vegetables, a heated brine solution is added at the filling stage, which generally consists of salt, sugar, and water. After washing, fruits may be cored and/or peeled, depending on the variety, and washed again to remove peeling residues. Fruits are then subjected to size reduction to obtain the desired form. Some canned fruit products, such as applesauce, are then cooked. Heated syrup or fruit juice is often added to fruits at the filling stage. After filling, the canned fruits and vegetables are exhausted, sealed, sterilized, and cooled before proceeding to final packaging operations.Figure 3.2 depicts representative process flows for the combined manufacture of canned diced tomatoes and canned tomato juices, pastes, and sauces. After inspection and grading, tomatoes are typically washed in a series of agitated water flumes. Next, color sorting is done either manually or automatically to remove green tomatoes, which are subsequently sent to pulping. The red tomatoes are then subjected to steam peeling, followed by manual sorting to remove tomatoes that have not been sufficiently peeled, hydroponic gutter which are also sent to pulping. Peeled red tomatoes are then diced and filled into cans using rotary brush fillers.The canned diced tomatoes are then exhausted, sealed, sterilized, and cooled before proceeding to final packaging operations.

The pulper is used to crush green and unpeeled tomatoes as well as pulping waste from the dicer. After pulping, the tomato slurry proceeds to the evaporator for concentration into juice, puree, and paste . Tomato purees are then typically mixed with other ingredients to create tomato sauce. Prior to filling, evaporated tomato products undergo continuous sterilization. Once filled the canned tomato juices, pastes, and sauces are sent to final packaging operations.The typical processing steps involved in fruit juice canning are depicted in Figure 3.3. After inspection, grading, and washing, juices are extracted from the fruits using mechanical expression or extraction methods. The juice is then often filtered to remove unwanted pulp, deaerated to remove excess oxygen, and deoiled. Next, the juice is pasteurized in a continuous fashion. For fresh juice manufacture, the pasteurized juice is immediately cooled and filled into a container before proceeding to final packaging operations. For canned juice manufacture, the pasteurized juice is hot filled into a container, which is subsequently exhausted, sealed, sterilized, and cooled before proceeding to final packaging operations.Energy represents a significant operating cost to the U.S. fruit and vegetable processing industry. In 2002, the industry spent nearly $810 million on purchased fuels and electricity, or roughly 4.5% of the industry’s total cost of materials . Of this, $370 million was spent on purchased electricity and $440 million was spent on purchased fuels . Electricity is used throughout the typical fruit and vegetable processing facility to power motors, conveyors, compressed air systems, and pumps, as well as building lighting and heating, ventilation, and air conditioning systems . Another major end use of electricity in the industry is refrigeration, which is used for process cooling, cold storage, and freezing applications. For all end uses, the U.S. fruit and vegetable processing industry consumed a total of 6.7 terawatt-hours of electricity in 2002, or nearly 10% of the electricity consumed by the entire U.S. food industry .

The major end use of fuels in the typical fruit and vegetable processing facility is in boiler systems for the generation of steam, which can be used in a wide variety of process heating, water heating, and cleaning applications . Although coal, residual oil, and distillate oils are sometimes used as fuels , currently natural gas accounts for over 90% of all fuels consumed by the U.S. fruit and vegetable processing industry . Thus, in discussions of both the end uses of fuels and the energy efficiency opportunities available for fuels in U.S. facilities, the remainder of this Energy Guide focuses exclusively on natural gas.In 2002, the U.S. fruit and vegetable processing industry consumed around 6.7 TWh of electricity, which equates to roughly 23 trillion Btu of final energy . The frozen fruit, juice, and vegetable manufacturing sub-sector was the industry’s largest consumer of electricity—due in large part to its extensive use of electricity for refrigeration—accounting for roughly 45% of the total electricity consumed by the industry in 2002. The fruit and vegetable canning sub-sector was the next largest user of electricity , followed by the dried and dehydrated food sub-sector and the specialty canning sub-sector . At least half of the industry’s electricity was expected to be consumed in the Western United States . The use of on-site electricity generation appears to be quite limited in the U.S. fruit and vegetable processing industry. In 2002, only 5% of the industry’s electricity was generated at individual facilities . The use of on-site generation was confined almost exclusively to the fruit and vegetable canning sub-sector, where the extensive use of steam in blanching, evaporating, pasteurizing, and sterilizing applications makes combined heat and power systems particularly attractive. The U.S. fruit and vegetable processing industry consumed an estimated 78 TBtu of natural gas in 2002.

The fruit and vegetable canning sub-sector was the industry’s largest consumer of natural gas, accounting for nearly one half of all industry natural gas consumption in 2002 . The frozen fruit, juice, and vegetable manufacturing sub-sector was the next largest user of natural gas, consuming an estimated 21 TBtu of natural gas in 2002, followed by the dried and dehydrated foods manufacturing subsector and the specialty canning sub-sector . At least one half of the industry’s natural gas was expected to be consumed in the Western United States . Table 4.1 summarizes the electricity and natural gas use of the U.S. fruit and vegetable processing industry. In total, the industry consumed an estimated 101 TBtu of final energy in 2002. Combined, the fruit and vegetable canning sub-sector and frozen fruit, juice, and vegetable manufacturing sub-sector accounted for around 75% of the industry’s total final energy use. Figures 4.4 and 4.5 depict the end uses of energy in these two important sub-sectors.The energy consumed by steam-based processes at individual canneries depends heavily on the type of equipment employed, the product manufactured, and equipment configurations. For example, steam blanchers have been reported to consume anywhere from 0.37 kg steam/kg product to 0.94 kg steam/kg product . Water blanchers have been reported to consume anywhere from 0.22 kg steam/kg product to 0.52 kg steam/kg product . Another major consumer of energy is the washing of incoming fruits and vegetables, which, depending on the facility, can use either hot water or ambient water and generally involves a high degree of mechanical agitation. For washing systems that use hot water, water efficiency measures and measures for recovering energy from hot water can be key strategies for reducing process energy consumption. For further details on water efficiency, see Chapter 15 of this Energy Guide. Table 4.3 shows energy intensity data for key processes used in juice canning. The two washing operations—incoming product washing and container washing—are seen to be the most energy-intensive processes involved, together consuming 434 Btu/lb of hot water. Thus, as for fruit and vegetable canning, water efficiency and heat recovery are likely to be key energy saving strategies in juice canning. The pasteurization process is the most significant consumer of steam, followed by the heat sterilization process. Tables 4.2 and 4.3 suggest that for most canneries, steam and hot water represent by far the most dominant uses of process energy in the facility, while process electricity use is generally of lesser significance.Representative process energy intensities for frozen fruit manufacture are provided in Table 4.4. As for canneries, hydroponic nft channel the processes of washing and blanching are likely to be the largest consumers of steam in a typical fruit freezing facility. However, unlike canneries, it can be seen that electricity use is as significant as steam use in the facility, primarily due to the electricity intensity of the freezing process. While the energy intensity of freezing at individual plants can vary widely based on the technology employed—typical energy intensity values for freezing technologies range from 250 Btu/lb to 1,750 Btu/lb —in general, freezing will be the most energy intensive operation in fruit freezing facilities by a significant margin.Similarly, the freezing process is the most energy intensive operation in the manufacture of frozen French fried potatoes, as can be seen in Table 4.5. After freezing, the next largest consumer of energy in frozen French fried potato manufacture is typically the frying process, which consumes a significant amount of direct fuel to heat the frying oil. Table 4.6 provides representative process energy intensity data for the manufacture of frozen concentrated citrus juice, one of the most significant product outputs of the U.S. fruit and vegetable processing industry . As in fruit freezing facilities, the freezing process accounts for the largest share of electricity use in frozen concentrated juice manufacturing facilities. However, the concentration process is the most energy intensive process by a significant margin, consuming an estimated 900 Btu of steam per pound of citrus juice concentrate. Thus, in addition to freezing, the concentration process is likely to be one of the most attractive opportunities for energy efficiency in the typical frozen concentrated juice facility.Lastly, representative process energy intensity data for dehydrated mashed potato manufacture are provided in Table 4.7. Peeling, precooking, and cooking are estimated to be very energy intensive processes. However, the most energy intensive process by far is the drum drying process, which consumes an estimated 6,000 Btu of steam per pound of dehydrated mashed potatoes. In fact, the drying process is one of the most energy intensive processes employed in the entire U.S. food processing industry, with typical energy intensity values ranging from around 1,500 Btu per pound of water in the product to over 28,000 Btu per pound of water in the product .Many opportunities exist within U.S. fruit and vegetable processing facilities to reduce energy consumption while maintaining or enhancing productivity. Ideally, energy efficiency opportunities should be pursued in a coordinated fashion at multiple levels within a facility. At the component and equipment level, energy efficiency can be improved through regular preventative maintenance, proper loading and operation, and replacement of older components and equipment with higher efficiency models whenever feasible. At the process level, process control and optimization can be pursued to ensure that production operations are running at maximum efficiency. At the facility level, the efficiency of space lighting, cooling, and heating can be improved while total facility energy inputs can be minimized through process integration and combined heat and power systems, where feasible. Lastly, at the level of the organization, energy management systems can be implemented to ensure a strong corporate framework exists for energy monitoring, target setting, employee involvement, and continuous improvement. The remaining chapters in this Energy Guide discuss some of the most significant energy efficiency measures applicable to fruit and vegetable processing at the component, process, facility, and organizational levels. This focus of this Energy Guide is on energy efficiency measures that are proven, cost effective, and available for implementation today. Whenever possible, measure descriptions include case studies of fruit and vegetable processing plants that have successfully implemented the measure, both in the United States and abroad. Many case studies include specific energy and cost savings data as well as typical investment payback periods. For measures where data are not available for fruit and vegetable processing facilities, this Energy Guide presents case study data from other sub-sectors of the food industry and occasionally from non-food industries to illustrate typical measure savings. Lastly, for most measures references to the technical literature and online resources are provided, which can be consulted for further information. For individual fruit and vegetable processing facilities, the actual payback period and savings associated with a given measure will vary depending on facility activities, configuration, size, location, and operating characteristics. Thus, the values presented in this Energy Guide are offered as guidelines.

AtbZIP11 transcript levels are upregulated by both light and sugars, which contribute to photoassimilates availability as a result of photosynthesis. When carbohydratesupply is sufficient, sucrose-mediated repression of AtbZIP11 translation would be initiated. In this way, AtbZIP11 activity could keep carbohydrate homeostasis in plant . In contrast, the expression levels of AtbZIP1 and AtbZIP53 were induced after extended night treatment and repressed by sugars application . In strawberry, bZIP11 was induced by red and blue light, while bZIP53 homologue was depressed. In addition, sucrose treatment did not significantly affect bZIP53 at transcriptional level . These findings indicated that bZIP S1 members differentiated in response to some factors. All of bZIP S1 transcriptional factors have the SIRT-responsive uORFs, so researchers proposed a novel SIRT-bZIP technology to enhance sweetness especially for some plant species rich in sucrose . In our study, strawberry bZIP11 overexpressing in tomato indeed increased the TSS and SS content and SS/TA ratio, which provided an applicable method for improvement of strawberry and other fruit quality in the future. However, constitutive overexpression of FvbZIP11 caused a growth impairment, which have been observed in tobacco , Arabidopsis , banana . An explanation of this phenotype was that bZIP11 presumably severely affected carbohydrate partitioning via a mechanism that might include direct regulation to cell-wall invertase and sucrose transporter expression . To avoid growth impairment, Sagor et al. overexpressed SlbZIP1 and SlbZIP2 under the control of the fruit-specific E8 promoter. The growth and morphology of the resulting transgenic tomato plants were comparable to those of wildtype plants. Most fruit-specific promoters currently available have been isolated from tomato, but these promoters probably are inappropriate to be used in non-climacteric fruits, like strawberry . Hence, round planter pot identification of a suitable promoter could facilitate the function analysis of a specific gene involving fruit development and help to specifically improve fruit quality.

The rise of agriculture c. 7000 BC ensured a stable food supply, allowing human civilizations to develop and populations to grow . The challenge of feeding a growing population is exacerbated by climate unpredictability, with drought and temperature increases, leading to decreased crop yield . Tomato is by far the most widely grown vegetable crop worldwide . The narrow genetic base of most crops, combined with selection for performance under optimal conditions, has reduced the genetic variability in environmental stress responses, and the modern cultivars of tomato are no exception . The wild relatives of tomato have the genetic ability to adapt to extreme habitats, and many heirloom cultivars also retain this ability as a result of directed breeding with wild species, and less selection for commercially valuable traits . Heirloom tomatoes are defined as varieties, which have been passed down through multiple generations of a family . Improvement in tomato has focused on flowering, fruit traits, and disease resistance probably as a result of a perceived negative correlation between fruit size and sugar content . Thus, potential impacts of other factors on yield and fruit quality are relatively ignored . In a previous study by Chitwood et al. , a meta-analysis on a set of introgression lines linked leaf complexity and leaflet shape in tomato to fruit sugar content measured on the same lines by other researchers . This correlation showed that plants with complex and rounder leaflets also had increased fruit sugar content . Because leaves are the primary site of photosynthesis, it is possible that leaf shape changes may impact photosynthetic capacity and therefore result in different sugar content and yield in fruits. In addition to photosynthesis, sugar transport, and distribution to sinks are other potential sites of regulation in leaf function as source tissue. While sugar transport in plants is well described, distribution among different sink tissues is not fully understood .

We analyzed tomato cultivars with varied yield and fruit quality, photosynthetic capacity, leaflet shape, and other vegetative traits and found that leaflet shape was strongly correlated to overall fruit quality assessed as a composite measure of BRIX and yield , with rounder leaflets positively correlated with higher BY values. Photosynthesis, on the other hand, had a negative correlation with yield. Based on our analysis, leaf shape seems to play an important role in the distribution of photo assimilates. Additionally, we performed phylogenetic network analysis on 23 cultivars, including eight identified as having the rounder Potato Leaf Morph , known to be caused by a mutation in the C-locus , to determine their breeding histories and identify any potential selection for this trait.Eighteen heirloom tomato varieties identified as having a range of fruit types, including cherry and beefsteak tomatoes, and several intermediate types, were analyzed. These tomato varieties also differed in fruit production timing from early to late, and the type of leaf morphology. These cultivars were selected based on leaf shape as described in Tatiana’s TOMATO base and The Heirloom Tomato . Tomato seeds were treated, germinated, and field planted as previously described . In both the 2014 and 2015 seasons, plants were laid out in a randomized block design and were planted and grown in soil, with furrow irrigation once weekly.Gas exchange measurements were done in the field on attached leaves after the plants had recovered from transplanting. Measurements were made weekly from week 10 to week 15 , on week 17 , and weeks 18– 21 , on c. 60 plants each week, on three plants per cultivar wk–1 . Measurements were made on leaves from the upper and lower portions of the plants to eliminate positional bias within the plant, and measured for three leaves per plant. The A , gst , transpiration, and ɸPS2 of a 6 cm2 area of the leaflet were measured using the LI-6400 XT infrared gas exchange system , and a fluorescence head .

Light within the chamber was provided by the fluorescence head at 1500 µmol m2 s 1 photosynthetically active radiation , and the chamber air flow volume was 400 µmols s 1 with the chamber atmosphere mixed by a fan. CO2 concentration within the chamber was set at 400 µmols mol 1 . Humidity, leaf and chamber temperature were allowed to adjust to ambient conditions; however, the chamber block temperature was not allowed to exceed 36°C. Measured leaflets were allowed to equilibrate for 2–3 min before measurements were taken, allowing sufficient time for photosynthetic rates to stabilize with only marginal variation. The amount of intercepted PAR was measured in four orientations per plant and an average PARi calculated. PARi was measured by placing a Line Quantum Sensor onto a base made from ¼” PVC piping, and a Quantum Sensor approximately 1 m above the plant on the PVC rig. Measurements from both sensors were taken simultaneously for each sample using a Light Sensor Logger . This allowed variation in overall light intensities such as cloud movement to be measured and accounted for in the total PARi.After gas exchange measurements, three plants per cultivar were destructively harvested each week. The final yield and fresh vegetative weight of each plant harvested was measured using a hanging scale in the field. Five leaves were collected at random from the bottom and top of the plant to capture all canopy levels, round pot for plants and approximately nine fruit were collected for BRIX measurements. FW was used owing to the large number of plants and measurements being done in situ in the field setting. All measurements were made in kg. To measure the BRIX value of the tomatoes, the collected fruit was taken to the laboratory where the juice was collected and measured on a refractometer . The yield and BRIX for each plant were multiplied together to get the BRIX 9 yield index , which gives an overall fruit quality measure, accounting for variations and extreme values in either measurement. It should be noted that while BRIX is used as a standard quality measure, BY is a composite value that folds in yield to assess weight of soluble solids per plant and is being used to measure commercial quality and not consumer quality . BY measurements were done for both the 2014 and the more detailed 2015 fields. These data were compared to test for reproducibility of results .The leaf complexity measures included all leaflets present on the leaf. Subsequently, primary leaflets were used for imaging and analysis of shape and size as previously described , and the images then processed in IMAGEJ . The images were cropped to individual leaflets maintaining the exact pixel ratio of the original image, and then cropped again to only include the single leaflet using a custom Java script written for FIJI . Single leaflet images converted to a binary image as black on a white background, and smoothed to allow for the exclusion of any particulates in the image were then processed in R using MOMOCS, a shape analysis package. Leaflet images were imported and then aligned along their axes so that all images faced the same direction. They were then processed using elliptical Fourier analysis based on the calculated number of harmonics from the MOMOCS package. Principal component analysis was performed on the resulting eFourier analysis and the principal components were used for subsequent analysis. Traditional shape measures such as leaflet area, circularity, solidity, and roundness were done with the area measurement based on pixel density. These measures were compared with the PCs to determine the characteristics captured by each PC. The PC values were used for all subsequent leaflet shape and size analyses. Total leaf area for each plant was measured by imaging the whole plant and a 4 cm2 red square and then processed in the EASY LEAF AREA software .Five plants per line were used to analyze leaflet sugar content. The plants were grown under the same conditions as field plants with the following exceptions. Plants remained in the glasshouse after transfer to 1 gallon pots. All plants were watered with nutrient solution and grown until mature leaves could be sampled. Using a hole punch, a disk with an area of 0.28 cm2 was taken from the leaflets and extracted from the disks using a modi- fied extraction method from the Ainsworth laboratory . Leaf disks were placed in 2 mM HEPES in 80% EtOH and heated to 80°C for 20 min and the liquid collected and stored at placed in 2 mM HEPES in 50% EtOH and heated, collecting the 20°C. The entire process was repeated twice. They were then liquid and storing at 20°C followed by another 2 mM HEPES in 80% treatment. The collected liquid was then used to measure the amount of sugar present per area of disk. To measure leaf sugar content a working solution of 100 mM HEPES , 6.3 mM MgCl2 , and 3 mM ATP and NADP at pH 7 was prepared. From the working solution, an assay buffer was made adding 50 U of glucose-6-phosphate dehydrogenase , and 295 or 280 µl of the working solution was added to a 96-well plate for sucrose standards or samples, respectively. Standards were added at a 60-fold dilution and samples were added at a 15-fold dilution. Then 0.5 U of hexokinase , 0.21 U of phosphoglucoisomerase , and 20 U of invertase were added to each well and the plates allowed to sit overnight to reach equilibrium. The plates were measured on a UV spectrometer at 340 nm, followed by analysis in JMP .All statistical analyses were performed using JMP software. To determine statistical significance, measurements were modeled using general linear regression model and tested by a one-way ANOVA followed by Tukey’s honestly significant difference, if necessary. These modeled data for all measured values were compiled into a table and used to create a model using partial least-squares path modeling in SMARTPLS 3.0 . Modeled data were used for the statistical analyses as many measurement types varied in number of data points, and therefore a set of generated predicted values of equal size was used to make an equal data matrix . Partial least squares-PM was used to explore the cause-and-effect relationships between the measured variables through latent values. PLS-PM is effective in both exploring unknown relationships and combining large-scale data, such as field, physiological, and morphological data, that otherwise are not well described together .

Our analysis of ripening-related gene expression in Cnr showed striking similarities to WT in the number and functions of genes changing between stages. Moreover, 69.5% of ripening related DEGs in Cnr were shared with WT . These results further support the hypothesis that Cnr is not exclusively a ripening mutant. Instead, Cnr fruit undergoes gene expression changes consistent with WT “ripening.” However, the ripening related changes in gene expression that occur in Cnr are not enough to compensate for the large defects accumulated in the fruit during growth and maturation. In a recent report, a knockout mutation to the gene body of CNR yielded little visible effects on fruit development and ripening , which suggests that the Cnr mutant phenotype may result from more than just a reduced expression of the CNR gene as previously reported . It has also been demonstrated that Cnr fruit have genome-wide methylation changes that inhibit ripening-related gene expression . The developmental defects observed in Cnr are likely caused by these methylation changes, directly or indirectly caused by the Cnr mutation . Thus, to better understand the Cnr mutation, more physiological data at earlier stages of development needs to be analyzed and complemented with more in-depth functional analysis of gene expression alterations at the corresponding stages. In addition, further molecular and genetic studies need to be performed and compared against complete CNR knockout mutants. Our data support that the mutants never produce a burst in ethylene production, even at the OR stage where more ripening phenotypes are observed . The orange-red pigmentation in nor OR fruit and the similarities of rin OR fruit in texture and taste-related attributes to WT RR fruit occur independently of an ethylene burst. These observations evidence that other regulatory mechanisms exist to initiate ripening events outside of ethylene .

Unlike previous reports, our data consistently showed that Cnr presented increased ethylene levels at the MG stage compared to WT . Interestingly, Cnr fruit produced more of the ethylene precursor ACC than WT at the RR stage. Also, rin made equivalent levels to WT fruit. Ethylene biosynthesis is divided into two programs: System 1 produces basal levels of the hormone during development, black plastic plant pots and System 2 generates the climacteric rise in ethylene during ripening . Each of these systems is catalyzed by a different set of ethylene biosynthetic enzymes . It is clear that all mutants show defects to System 2 of ethylene biosynthesis, but they also appear to have alterations specific to System 1. For example, we observed that SlACO3, a System 1- specific ACC oxidase, was higher expressed in Cnr fruit than WT .The role of ABA in climacteric ripening is not as well explored but has been reported to be complementary to ethylene . Previous reports in WT fruit have shown that ABA increases until the breaker stage, just before the ethylene burst . ABA has also been shown to induce ethylene production and linked to the NOR transcription factor . We found that nor and rin fruit did not show decreases in ABA concentration during ripening like WT did . For nor, the constant levels of ABA between MG and RR stages are another example of how fruit ripening events are delayed or inhibited. RIN and ABA have been demonstrated to have an inverse relationship where RIN expression is repressed with the induction of ABA . The significant increase of ABA accumulation in rin during ripening suggests that ABA biosynthesis and metabolism are misregulated in this mutant. rin fruit appear to present a delayed peak in ABA levels compared to WT fruit. Our results support the indirect interaction between the TFs and ABA during ripening. More developmental stages, genetic manipulations, and exogenous hormone treatments are needed to investigate further the trends of ABA accumulation seen in the ripening mutants.

The interactions between the CNR, NOR, and RIN in ripening have been debated in the literature . The TF RIN directly interacts with NOR and CNR, binding to their respective promoters, and therefore has been proposed to be the most upstream TF among the three regulators . Here we provided evidence that the three TFs display at least indirect effects on each other. We have argued that the Cnr mutant shows a wide breadth of defects across fruit development before ripening begins, and thus, we propose the Cnr mutation is acting before NOR or RIN. This further supports the hypothesis made in Wang et al. that Cnr acts epistatically to nor and rin. The gene expression patterns of CNR, NOR, and RIN across ripening stages were decreased or delayed in each of the single ripening mutants. The most substantial variation in gene expression was the downregulation of NOR and RIN expression across all stages in the Cnr mutant . We present for the first time double ripening mutants, homozygous for both loci, that can be used to see the combined effects of each mutation on fruit development and quality traits. We successfully generated the double mutants by establishing reliable and high throughput genotyping protocols for each mutation and evaluating segregation of the mutant phenotypes in field trials across multiple growing seasons. We obtained double mutants from both reciprocal crosses but saw no fruit phenotypic differences between them, suggesting that the ripening mutations are not influenced by maternal or paternal effects . Because the nor and rin mutants look so similar, it was hard to visually determine the individual effects of each mutation on the appearance of rin/nor fruit. However, when specific fruit traits were measured, we could detect additive or intermediate fruit phenotypes in this double mutant, supporting the proposed relationship in Wang et al. . Thus, nor and rin appear to influence similar fruit traits and act in coordination.

The Cnr mutation had a significant effect on the Cnr/nor and Cnr/rin mutants resulting in fruit with similar appearance and ethylene production to the Cnr fruit . When analyzing the gene expression profiles of the Cnr/nor fruit, we also observed multiple similarities to the Cnr parent, but also several deviations . Surprisingly, Cnr/nor was also reminiscent of nor, as it displayed few ripening-related gene expression changes, suggesting the inhibition or delay of specific ripening events in nor carried over to the double mutant. Here, we proposed that the Cnr mutation causes defects throughout fruit development while the nor mutation causes defects predominantly in ripening. However, the Cnr/nor double mutant showed additional phenotypic and transcriptional defects before ripening than both mutant parents . These observations indicate that in combination with Cnr, nor may contribute to alterations in early fruit development and the inhibition of ripening progression.Fruit breeders actively selected several morphological and quality phenotypes during the domestication of the garden strawberry , an allo-octoploid of hybrid origin. F. × ananassa was created in the early 1700s by interspecific hybridization between ecotypes of wild octoploid species , multiple subsequent introgressions of genetic diversity from F. virginiana and F. chiloensis subspecies in subsequent generations, and arti-ficial selection for horticulturally important traits among interspecific hybrid descendants. Domestication and breeding have altered the fruit morphology, development, and metabolome of the garden strawberry, distancing modern cultivars from their wild progenitors. Approximately 300 years of breeding in the admixed hybrid population has led to the emergence of high yielding cultivars with large, firm, visually appealing, long shelf life fruit that can withstand the rigors of harvest, handling, storage, and long-distance shipping. Fruit shape is an essential trait of agricultural products, particularly those of specialty crops, owing to perceived and realized relationships with the quality and value of the products. Image-based fruit phenotyping has the potential to increase scope, throughput, and accuracy in quantitative genetic studies by reducing the effects of user bias, enabling the analysis of larger sample sizes, and more accurate partitioning of genetic variance from environments, management, and other non-genetic sources of variation. Many fruit phenotyping approaches rely on the human eye to sort fruit into discrete, descriptive categories for planar shapes. Categories are either nominal, existing in name only, or ordinal, referring to a position in an ordered series or on a gradient. Classification into categories is often labor-intensive and prone to human bias, black plastic garden pots which can increase with task complexity and time requirements. Alternative scoring approaches rely on morphometrics and machine learning to automate classification; e.g., sorting fruit into shape categories in both tomato and strawberry. Unsupervised machine learning methods , unlike supervised methods, are useful for pattern detection and clustering, while supervised machine learning methods are useful for prediction and classification.

Unsupervised clustering enables the calculation of several measures of model performance and overfitting to balance compression and accuracy. However, the categories derived from these techniques are without order, resulting in the need for a suitable transformation to an ordinal scale more appropriate for quantitative genetic analyses. In this context, ordinal categories give the interpretation of relationship with, or distance from, other shape categories in a series. To enable this interpretation, we developed a method for asserting the progression through fruit shape categories derived from unsupervised machine learning methods. The Principal Progression of k Clusters allowed us to nonarbitrarily determine the appropriate shape gradient for statistical analyses using empirical data. The advantages of PPKC, relative to a manually determined ordinal scale, are that it does not require arbitrary, a priori decisions and is unsupervised, which obviates additional operator bias. Here, we describe approaches for translating digital images of strawberries into computationally defined phenotypic variables for identifying and classifying fruit shapes. Fruit shape and anatomy are complex, multi-dimensional, and, potentially, abstract phenotypes that are often not completely or intuitively described by planar descriptors and individual qualitative or quantitative variables. Beyond the qualitative definitions used in plant systematics, references to fruit shape encompass a wide variety of mathematical parameters and geometric indices that establish quantitative measurements of plant organs . Much like human faces or grain yield, fruit shape and anatomy are products of the underlying genetic and non-genetic determinants of phenotypic variability in a population. Quantitative phenotypic measurements have allowed researchers to uncover some of the genetic basis of fruit shape in tomato, pepper, pear, melon, potato, and strawberry. However, the major genetic determinants of fruit shape remain unclear, or understudied, in octoploid strawberry, in part because researchers have not yet translated fruit shape attributes into holistic, quantitative variables, which may empower the identification of underlying genes or quantitative trait loci through genome-wide association studies and other quantitative genetic approaches. Quantitative features often rely on linear metrics of distance and are generally modified into compound descriptors that remove the effects of size. However, compound linear descriptors often have limited resolution compared to more comprehensive, multivariate descriptors. Elliptical Fourier analysis quantifies fruit shape from a closed outline by converting a closed contour into a weighted sum of harmonic functions . Generalized Procrustes analysis quantifies the distance between sets of biologically homologous, or mathematically similar, landmarks on the surface of an object. Fruit shape can also be described using linear combinations of pixel intensities from digital images extrapolating from analyses generally used to quantify color patterns and facial recognition. Similar pixel-based descriptors have recently been referred to as ”latent space phenotypes” and arise from unsupervised analyses that allow a computer to produce novel, independently distributed features directly from images. Here, we generate a dictionary of 68 quantitative features, including linear-, outline-, landmark-, and pixel-based descriptors to investigate the quality of different features in preparation for quantitative genetic analyses. The ultimate goal of our study was to develop heritable phenotypic variables for describing fruit shape, which could then be used to identify the genetic factors underlying phenotypic differences in fruit shape. The phenotyping and analytic workflow for this study are summarized in Figs 1 and 2. We first describe and demonstrate the application of PPKC, which transforms categories discovered from unsupervised machine learning methods to a more convenient and analytically tractable ordinal scale. We then explore the relationship between machine acquired categories and 68 quantitative features extracted from digital images. Next, we apply random forest regression to select critical sets of quantitative features for classification and use supervised machine learning methods, including support vector regression and linear discriminant analysis , to determine the accuracy of shape classification.

Finetuning of metabolomic traits such as amylose content in rice and sugar content in wild strawberry recently were made possible via CRISPR-Cas9 gene-editing technology. Similar approaches can be taken in cultivated strawberry for flavor improvement, but not before the biosynthetic genes responsible for metabolites production and their regulatory elements are identified. Our pipeline has proven to be effective in identification of novel causal mutations for flavor genes responsible for natural variation in volatile content and can be further applied to various metabolomic and morphological aspects of strawberry fruit such as anthocyanin biosynthesis , sugar content and fruit firmness. These findings also will help breeders to select for genomic variants underlying volatiles important to flavor. New markers can be designed from regulatory regions of key aroma volatiles, including multiple medium-chain volatiles shown to improve strawberry flavor and consumer liking , methyl thioacetate contributing to overripe flavor and methyl anthranilate imparting grape flavor . In the present study, a new functional HRM marker for mesifurane was developed and tested in multiple populations . These favorable alleles of volatiles can be pyramided to improve overall fruit flavor via marker assisted selection. Specific esters are shared with apple , certain lactones are shared with peach and various terpenes are shared with citrus . Syntenic regions and orthologous genes could be exploited for flavor improvement in those species. Additional insights were gained for the strawberry gene regulatory landscape, SV diversity, complex interplays among cis- and trans- regulatory elements, and subgenome dominance. Previously, Hardigan et al. and Pincot et al. showed a large genetic diversity existing in breeding populations of Fragaria × ananassa, plastic grow pots challenging previous assumptions that cultivated strawberry lacked nucleotide variation owing to the nature of its interspecific origin and short history of domestication .

Our work corroborated their findings and showed that even highly domesticated populations harbor substantial expression regulatory elements and structural variants. Over half of the expressed genes in fruit harbored at least one eQTL, and 22 731 eGenes had impactful cis-eQTL. The distribution of trans-eQTL is not random, but rather is concentrated at a few hotspots controlled by putative master regulators . The aggregation of trans-eQTL also was observed in plant species such as Lactuca sativa and Zea mays . Furthermore, we observed a substantial number of trans-eQTL among homoeologous chromosomes, similar to observations in other allopolyploid plant species . In cotton, physical interactions among chromatins from different subgenomes have been identified via Hi-C sequencing , supporting a potential regulatory mechanism among homoeologous chromosomes. However, owing to the high similarity among four subgenomes and limited length of Illumina reads, false alignment to incorrect homoeologous chromosomes could arise, leading to ‘ghost’ trans-eQTL signals. Future studies are needed to scrutinize the homoeologous trans-eQTL and investigate the mechanism behind this genome-wide phenomenon. Higher numbers of trans-eQTL in the Fragaria vesca-like subgenome are consistent with its dominance in octoploid strawberry . By contrast, the highly mixed Fragaria viridis- and Fragaria nipponica- like subgenomes contained much smaller numbers of trans-eQTL. The characterization of naturally-occurring allelic variants underlying volatile abundance has direct breeding applications. First, this will facilitate the selection of desirable alleles via DNA markers. Second, understanding the causal mutations in alleles can guide precision breeding approaches such as gene editing to modify the alleles themselves and/or their level of expression. From a broader perspective, multi-omics resources such as this one will have value for breeding a wide array of fruit traits.

Enhancing consumer satisfaction in fruit ultimately will depend on the improvement of the many traits that together enhance the overall eating experience.In a fruit tree orchard system, individual trees are composed of two genetically different genotypes, one being the rootstock which includes the mass of the tree below the soil surface to a graft union about midway up the trunk. Rootstocks can be selected for pest resistance or tolerance towards adverse soil conditions, and they can also influence vigor and cropping . The second portion of the tree is referred to as the scion and accounts for most of the above-ground mass, usually chosen for fruit production traits . Over the last 40+ years, the University of California has had a peach rootstock development program that has identified several promising size controlling rootstocks which allow for the establishment of new commercially viable orchard systems . New dwarfing rootstocks for peach must be graft compatible, reduce vigor, and not diminish marketable fruit production by reducing fruit size or quality . Previous peach rootstock trials monitored vigor control and grafting compatibility in conventional planting systems however, yield parameters such as fruit size and quantity have not been as thoroughly evaluated using these rootstocks in pedestrian orchard systems . Fruit size is paramount in peach production as larger fruit, free of cosmetic imperfections, have a higher market demand and therefore higher market value . It has been reported that peach fruit produced on trees with size-controlling rootstocks can tend to be smaller in size than fruit on trees with more vigorous rootstocks .Vascular tissue known as xylem is responsible for the movement of water and nutrients in all trees. In trees, every year a new ring of xylem forms surrounding the previous year’s growth and water conduction in the xylem often occurs only in this outermost annual ring . It has been reported that size-controlling peach rootstocks contain a higher proportion of narrow diameter xylem vessels and fewer larger vessels when compared to more vigorous rootstocks in addition to having an increased axial diameter .

Both characteristics create a reduction of hydraulic conductance in the size-controlling peach rootstocks compared to traditional, vigorous rootstocks. Reduced hydraulic conductance, as demonstrated by and , can cause reductions in stem water potential during mid-day hours that can lead to a reduction in vegetative growth .An mean peach fruit’s fresh weight is composed of over 80% water . Thus, it is reasonable to assume a reduced hydraulic conductance created by size controlling rootstocks could hinder fruit size. However, the relationship between fruit growth and water availability is dynamic and depends on the developmental stage of the fruit, the severity of water limitations, and the component of growth being considered . It has been reported that mild water stress applied during the intermediate developmental period of slow fruit growth has no effect on crop yields but significantly reduces vegetative growth in peach . Fruit developmental stages may differ in time of initiation and duration among peach varieties, an example of this would be an early vs. late harvested cultivar as demonstrated by . Fruit growth occurs in stages from fruit set to harvest, in all cultivars, and during the final growth phase of peach fruit is when 65% of a fruit’s dry weight and 80% of a fruit’s fresh weight are accumulated . Available water varies throughout the growing season, including diurnal fluctuations brought on by daily temperature fluctuations , day-to-day changes brought on by a shift in evapotranspiration , and possible seasonal changes brought on by the formation of new xylem . Water conduction in the tree is largely dependent on newly formed xylem each spring and the new xylem cells are smaller in size-controlling rootstocks. It is thought that the spring flush of vegetative growth is limited in trees on size controlling rootstocks compared to growth on vigorous rootstocks because of temporary reductions in root hydraulic conductance caused by smaller xylem vessels. A question that arises from these findings, does the reduction of water conductance in dwarfed peach trees also limit fruit growth?In peach production, fruit size is often manipulated with the use of a management practice known as fruit thinning. With fruit thinning, shortly after fruit set, a portion of immature fruit is removed from the tree to reduce carbohydrate competition among those remaining. It is widely recognized that fruit size is largely influenced by crop load, with larger fruit size obtained as the crop load is reduced . Quality of fruit may also be affected by crop load, low-cropped trees have been shown to produce larger and firmer fruit than those from heavily cropped trees . Although minor in comparison tocarbohydrate demand, big plastic pots fruit size may also be diminished by inducing higher water stress with larger crop loads. An experiment by found that larger crop loads were responsible for reducing midday stem water potential in nectarines.

MacFayden et al., concluded that an increased crop load also increased the fruit water deficit which may reduce fruit growth in peach. According to another study by , rootstocks also influenced the crop load’s effect on fruit size, and more vigorous rootstocks had larger fruits at specific crop loads. The fore mentioned findings relay the importance of better understanding the relationship between fruit size and crop load among vigorous and reduced-vigor rootstocks.While crop load per tree is controlled by thinning, crop load per area is most influenced by planting density. The reduced vigor and overall size of trees on size-controlling rootstocks facilitates the establishment of high-density plantings . The primary principle in establishing an appropriate planting density for an orchard using trees on size controlling rootstocks is that total tree dry matter production and crop yield are related to total light interception . This principle holds for essentially all crops . However, although higher light interception often leads to higher yields, yield may also vary significantly with other environmental stressors such as available water, nutrients, temperature, and amount of time the fruit has for growth . Orchard systems with increased planting densities have also been shown to reach maximum yield capacity earlier than conventional plantings since the trees are able to fill out their allotted space more quickly . In a small trial using the ‘Summer Bright” nectarine cultivar, trees that were pruned to a standard height of 12 to 13 feet or limited to heights of 8 or 9 feet produced similar sized fruit and crop yields. The reasoning for this was that, despite the height difference, both tree shapes had equal planar volume and therefore intercepted similar amounts of photosynthetically active radiation .The goal of this study was to address three production characteristics and their relationship with four different orchard systems. 1) Fruit size: can peach orchard systems using trees on size controlling rootstocks produce fruit of equal size compared to orchard systems with trees on vigorous rootstocks? 2) Fruit count: if crop load per area is similar among size-controlling and vigorous systems is fruit size also similar? 3) PAR interception and yield: is there a difference in the relationship of fruit production vs light interception among orchard systems with vigorous rootstocks and those with size-controlling rootstocks? A better understanding of production capabilities will allow researchers and growers to better estimate the potential of an orchard system on size-controlling rootstocks as a commercially viable option.In April 2015, an orchard system trial was established at the University of California Kearney Agricultural Center, Parlier, CA. The research block consisted of two peach [Prunus persica Batsch] scion cultivars, June Flame and August Flame grafted onto three different rootstock genotypes: HBOK 27 , P-30-135 , and Nemaguard . Controller 6 was used in two of the four training systems . The C-6 V was a high-density planting system with an in-row spacing of 1.2m and trained to the KAC-V perpendicular V pruning system . The C-6 Quad system was pruned to a Quad V where four main scaffolds are selected in each tree and pruned to resemble an open vase, the system also had a larger in-row spacing of 2.4 m . The Controller 9 Quad system was identical to the C-6 Quad system with the only difference being the rootstock. Between-row spacing was 4.6m in all systems using size controlling rootstocks. Nemaguard was used as the commercial standard rootstock with a planting density of 2.4m in-row spacing and 5.5m between-row spacing . Shortly after harvest, orchard systems using size-controlling rootstocks were topped to a height of 2.5m while systems using the Nemaguard rootstock were topped at 3.5m. The four systems were divided into three replications for each of the two scion cultivars making a total of eight unique orchard systems. Each replication consisted of four rows of trees with the northern and southern most rows used as guard rows, the first and last two trees in each data row were also considered guard trees making nine trees in each of the two inner rows the sample size per replication . In total, each cultivar was represented by approximately 54 data trees .

Tertiary models are established based on primary and secondary models and use predicted values of growth parameters from secondary models to predict changes in pathogen density at times and levels of independent variables that have not been tested or used in the model development .The survival of all three pathogens was longest in high-moisture and low-moisture dates at refrigerated temperature. The combination of their high pH values and low aw compared to the other dried fruits may be reasons why the dates have larger D-value than the other dried fruits. Juneja et al found that L. monocytogenes, E. coli O157:H7, and Salmonella spp. were able to survive on dates for 32 days when stored at 4 °C. Furthermore they found that there was no significant difference in any of the pathogens when the dates were treated with antimicrobial washes of peracetic acid or with ethanol . Because of this strong ability for pathogens to survive, Medjool dates should be further explored from a microbial safety viewpoint. The storage temperature had the biggest influence on decimal reduction time in Salmonella. While having the highest D values of the three pathogens in refrigerated dried fruit, Salmonella had the lowest D values in the dried fruit stored at ambient temperature . This shows that temperature has a large influence on the survival time of Salmonella. While thermal death time does increase for all three pathogens when put in colder conditions, the difference in Salmonella is the starkest. For example, according to the models made, the time is would take to reduce Salmonella by 90% in high moisture dates at refrigerated temperature would be 396 days, whereas at ambient temperature it would take 21 days . These results suggest that Salmonella has particularly increased survival at lower temperatures compared to other pathogens. While the specific mechanisms that allow for this survival in low moisture environments are not completely clear, garden pots square temperature most likely has an influence on those mechanisms. Andino and Hanning suggest one possible mechanism that Salmonella spp. may use to enhance its survival at lowered temperatures is cold shock proteins .

Upregulation of these proteins allow Salmonella to adapt to colder environments as temperatures drop, leading to better survival of the pathogen. Looking at the dried peaches , the decimal reduction time of the pathogens were higher when the peaches were inoculated with the dry carrier versus the wet carrier. This suggests that pathogens are more persistent when using a dry carrier to simulate a dry environment. However, a factor that might have influenced the lower D value in the wet inoculated peach is that the initial inoculation strength is several logs higher with a wet carrier than a dry carrier. Due to the higher initial microbial load in the wet inoculated peaches compared to the dry inoculated, there is difficulty in comparing the true impact that the wet and dry carrier had on the decimal reduction time.Dried fruit were inoculated with Salmonella spp., Escherichia coli, or L. monocytogenes to determine how they would survive in two storage conditions. All three pathogens were able to survive in dried fruits and should be taken into consideration when looking at the safety of dried fruit processing. Salmonella had the longest survival potential among all three tested pathogens. This observation is expected as Salmonella is known for its ability to survive in low moisture conditions. The condition that allowed for longer pathogens survival was storage at refrigerated temperature rather than ambient temperature. This is important because many dried fruit processors store their dried fruits at refrigerated temperatures if not being sold immediately. This allows for a longer shelf life of the dried fruit compared to ambient storage, but increases the ability for bacterial survival. This may be due to the intrinsic factors of the dried fruit: pH, aw, and available nutrient. Based on the current data, survival was the longest in the dried fruits that had the highest relative pH and the lowest relative aw. Salmonella inoculated in Medjool dates survived to the very end of the 180-day survival study.

The Medjool dates had the highest pH of all the dried fruits and had some of the lowest aw of the dried fruits. Other intrinsic factors that were not measured could have also played a role in the long survival time in the dates. For instance, dates are known to have a high sugar content, which may have played a role in pathogen survival. Measuring various compounds in the dried fruits might give more insight on why certain dried fruits allowed for longer survival than others. Regardless the reason, pathogen survival was long in Medjool dates, and should be something that those who produce dates consider. Since dates are not dried the same way other dried fruits are, the steps in the date harvesting process should be looked at carefully. While conducting this research, a new potential outbreak associated with Medjool dates was reported . Twenty-eight people in England were infected with Hepatitis A in 2021 and is suspected to be from Medjool dates . The dates have since been recalled due to their possible contamination . In 2018 there was another outbreak of Hepatitis A in Denmark and was believed to be from dates from Iran . There has been no evidence to show that those dates were contaminated with the virus . That potentially makes this 2021 outbreak the first to be associated with a dried fruit not part of a mixed product. With the occurrence of this outbreak, it makes it all the more important to understand when and where potential contamination of pathogens in dried fruits can occur. Although the data generated from our study is based on bacteria, we did see that the survival of pathogens in Medjool dates is longer compared to other dried fruits. Additional research will be necessary to better understand the survival of foodborne virus on dried fruits. As discussed earlier in this thesis, there are many pre- and post-drying treatments that can be applied to fresh or dried fruits. When looking into the available literature, the efficacy of these treatments has not been systematically evaluated. One on-going project in the lab is to summarize the current knowledge about these treatments and their efficacies and develop a study that fills in the knowledge gaps. In the meantime, identifying a surrogate for testing the different pre- and post-drying treatments as well as different drying methods is needed.

Enterococcus faecium NRRL B-2354 has been validated and approved for being used as a surrogate for almond thermal processing validation . However, whether this strain can be as a surrogate for dried fruit related studies or not still needs further evaluation. One on-going test project in the lab is go evaluate the survival of E. faecium NRRL B-2354 in dried peaches and apricots. In this first test trial, E. faecium was inoculated onto two types of dried fruits and its survival at ambient and refrigerated temperatures is being monitored. In addition, the highest temperatures that can be achieved by various dry methods are being monitored and recorded. The efficacy of different pre-drying treatments is also being tested in the lab by using Salmonella-inoculated peaches and E. faecium-inoculated peaches. In summary, the microbial safety of dried fruits is important and needs more research attention. The survivability of common foodborne pathogens on different types of dried fruits and the recalls and outbreaks associated with dried fruits highlight the importance of the validation of pre- and post-drying treatments as well as different drying methods. The findings of this study, square pots along with future work, hopes to provide the foundation needed for the development of food safety plans for dried fruits.Under global warming and climate change, cultivated plants are encountering increased biotic and abiotic stresses, which lead to reductions of plant growth and reproduction and consequently economic losses. The use of plant endophytic bacteria to promote plant growth and increase tolerance of environmental stresses has provided an alternative to standard agricultural practices that has fewer safety concerns. Endophytic bacteria can be defined as non-pathogenic bacteria that colonize the interior of host plants and can be isolated from surface-sterilized plant tissues. These bacteria can obtain a constant nutrient supply from host plants by living inside the plants and having close contact with plant cells. The endophytic bacteria colonization process is usually initiated at wounds and cracks in the roots by a rhizospheric population of the bacteria in the soil. After entering the plant roots, endophytic bacteria can systemically colonize the above ground parts of plants, including stems and leaves.A wide diversity of endophytic bacteria has been discovered in several plant species. Endophytic bacteria communities include five main phyla. Proteobacteria is the most dominant phylum isolated from host plants, which includes α-, β-, and γ-Proteobacteria. Actinobacteria, Planctomycetes, Verrucomicrobia, and Acidobacteria are also commonly identified. The most frequently isolated bacteria genera are Bacillus, Burkholderia, Microbacterium, Micrococcus, Pantoea, Pseudomonas, and Stenotrophomonas, with the two major genera being Bacillus and Pseudomonas. Several factors affect the composition of endophytic bacteria populations, including plant growth conditions, plant age, types of analyzed plant tissues, soil contents, and other environmental factors. Endophytic bacteria can have several beneficial effects on host plants, such as promotion of plant growth and yield, increased resistance to plant pathogens, enhanced tolerance to abiotic stresses, elimination of soil pollutants through the facilitation of phytoremediation, and production of various metabolites with potential applications in agriculture, medicine, and industry. Some endophytic bacteria help host plants acquire increased amounts of limited resources from the environment. This can include enhancing the uptake of nitrogen, phosphorous, or iron by expressing nitrogenase, solubilizing precipitated phosphates, or producing iron-chelating agents in bacteria, respectively. Some endophytic plant-growth-promoting bacteria can increase host plants’ metabolism and nutrient accumulation by providing or regulating various plant hormones, including auxin, cytokinin, gibberellins, or ethylene. Auxin and ethylene are the two major hormones that affect plant growth and development and that are involved in plant-endophytic bacteria interactions. In addition to these four hormones, several endophytes can utilize signaling pathways mediated by salicylic acid, jasmonic acid, and ethylene to initiate induced systemic resistance and protect host plants from phytopathogen infection. A number of endophytic bacteria can also produce various antibiotics, toxins, hydrolytic enzymes, and antimicrobial volatile organic compounds to limit pathogen infection. We previously isolated a plant endophytic bacterium, Burkholderia sp. strain 869T2, from surface-sterilized root tissues of vetiver grass. Strain 869T2 can also live within banana plants, in which it promoted growth and reduced Fusarium wilt disease occurrence. Genomic sequences of the strain 869T2 contain the gene for 1-aminocyclopropane-1-carboxylate deaminase, which may modulate host plant ethylene levels. Strain 869T2 also has genes related to the synthesis of pyrrolnitrin, which may function as a broad-spectrum anti-fungal agent, as well as dioxin-degradation-related genes. Furthermore, strain 869T2 can degrade the toxic dioxin congener 2,3,7,8-tetrachlorinated dibenzo-p-dioxin , mainly via its 2-haloacid dehalogenase. A recent study compared the genome sequences of 31 Burkholderia spp. and reclassified Burkholderia cenocepacia strain 869T2 as Burkholderia seminalis. We also compared the genome sequences of the strain 869T2 with those of five published B. seminalis strains: FL-5-4-10-S1-D7, FL-5-5-10-S1-D0, Bp9022, Bp8988, and TC3.4.2R3. The strain 869T2 shared 93–95% of its genome with the other five B. seminalis strains. Furthermore, strain 869T2 lacked several genetic loci that are important for human virulence. Based on the results of our analysis of the core genome phylogeny and whole-genome average nucleotide identity , strain 869T2 was classified as B. seminalis. B. seminalis is a member of the Burkholderia cepacia complex , which is a group of Gram-negative, aerobic, non-sporulating, rod-shaped bacteria. Bcc consists of opportunistic human pathogens that exist in patients suffering from cystic fibrosis as well as pathogens of many vegetables and fruits, such as onion and banana. Contrary to the pathogenic traits that led to their original discovery, some Bcc bacteria have ecologically beneficial interactions with host plants. The plant endophytic bacterium B. seminalis strain TC3.4.2R3, isolated from sugarcane, can serve as a biocontrol agent to reduce infections with Fusarium oxysporum and the cacao pathogens Moniliophthora perniciosa , Phytophthora citrophtora, P. capsici, and P. palmivora as well as orchid necrosiscaused by Burkholderia gladioli through the production of pyochelin, a rhamnolipid, and other unidentified diffusible metabolites. Another strain of Burkholderia seminalis, strain R456 isolated from rice rhizosphere soils, decreased the occurrence of rice sheath blight disease caused by Rhizoctonia solani.