Month: January 2025

Rather than increasing the fluorescent signal in the deep cell layers, the entire triangular pattern of pWUS:eGFP-WUS shifted down by one cell layer. This occurred with little or no loss in fluorescent amplitude, though the slope of the upper gradient became slightly shallower in proportion to greater distance separating L1 cells from the new concentration peak.WUS proteins are known to occur in a nuclear localized pattern, which suggests involvement with nuclear pore trafficking mechanisms. To determine if cytokinin regulate WUS movements through the nuclear pore, two modified version of the pWUS:eGFP-WUS reporter were obtained, which contained nuclear import and nuclear export sequences tags: pWUS:NLS-eGFP-WUS, which was previously described by [20], and pWUS:eGFP-NES-WUS. If cytokinin favors nuclear import over export, or vice versa, one of these constructs should amplify the nuclear/cytoplasmic distribution of the fluorescent signal, producing a significantly different fluorescent pattern. As seen in Figure 3.6 however, the only significant change produced by 24 hour cytokinin treatments was a doubling of the fluorescent signal, which affected both modifiedreporters and the pWUS:eGFP-WUS control alike. Expression of pWUS:NLS-eGFP-WUS was more clearly nuclear localized than pWUS:eGFP-WUS as expected, while pWUS:eGFP-NESWUS produced very little fluorescent signal under either treatment. To more accurately estimate the nuclear/cytoplasmic ratio, these resulting confocal images were further analyzed in order to estimate the relative concentration of fluorescent molecules in each subcellular compartment. First the average fluorescent concentration was measured in a portion of each subcellular compartment, black plastic planting pots and these figures were then multiplied by the volume of the cytoplasm and nucleus, respectively.

Nuclei were assumed to be spherical, and their volume was calculated directly from the largest observable diameter. The volume of the whole cell was more difficult to obtain however, as optical sections often only allowed measurements of their length and width, as 3-D optical reconstructions frequently did not include the entire cell volume. Instead, “depth” was estimated using the average of the length and width measurements, reflecting the approximately cubic-rectangular shape of the cells in L1-L3. However, when compared to presumably more accurate cell volumes measured using a tessellation method, the volumes calculated by the present study were on average 2x larger than expected. The present volume estimates did not change significantly when “depth” values were substituted with measurements from unrelated SAM images , suggesting that these volume estimates are at least reasonably accurate, even if they lack precision. Curiously, the smallest volumes produced by the tessellation method closely approach the largest nuclear volumes obtained in the present analysis , raising the possibility that this computer-automated method may have occasionally measured nuclei and/or vacuoles instead of entire cells.Overall, pWUS:NLS-eGFP-WUS plants were found to produce about 15% smaller cells, 15% smaller nuclei, and 15% less total fluorescence when compared to pWUS:eGFP-WUS plants, but otherwise both reporters displayed similar subcellular distribution patterns: The L1 and L2 cells had identical nuclear/cytoplasmic ratios, and these values were independent of the total fluorophore concentration in each cell. In contrast, L3 cells had a distinctly elevated nuclear/cytoplasmic ratio that was on average almost twice as large as the upper two cell layers, and as much as 5x more in a outlier data. All nuclei held 2-4x more fluorescent units than would be expected based on their volume alone, yet counter-intuitively, this was just a small fraction of the total number of fluorescent units within the cell.

Instead, the majority of fluorescent units were found in the larger volume of the cytoplasm, though at a lower concentration than what occurs inside the nucleus. Cytokinin treatments did not significant change the nuclear/cytoplasmic ratios for either reporter, nor were any layer-specific patterns induced by this treatment. The only clear response to cytokinin treatment was a change in nuclear volume, which increased by an average of 154% in all three layers in both backgrounds. The change in nuclear volume apparently occurred at the expense of the cytoplasm, as the total cell volume remained constant . Unexpectedly, the pWUS:NLS-eGFP-WUS reporter was found to have nuclear/cytoplasmic ratios that was essentially identical to those produced by pWUS:eGFP-WUS. This is inconsistent with the idea that the NLS tag drives nuclear import, though analysis of the pWUS:NLS-eGFPWUS longitudinal gradient did find that protein movements into the L1 and L2 was slightly restricted , consistent with NLS trapping it inside the nucleus. However, this data also suggests an interesting paradox, as it implies that nuclear trapping occurs without significant nuclear enrichment.Another possible way in which cytokinin responses might affect the distribution of WUS protein is by regulating WUS translation and degradation rates. To study this possibility, the pWUS:eGFP-WUS reporter was exposed to the chemical inhibitors cyclohexmide and MG132, blocking protein translation and proteosome-mediated decay, respectively. Following 4 hour treatments with cycloheximide, no significant loss of fluorescence was found. Unexpectedly, the comparable treatment with MG132 led to the rapid loss of the fluorescent signal.

When these chemical treatments were supplemented with exogenous 6-benzylaminopurine to boost the cytokinin response above a basal level however, these patterns were completely reversed.The absence of cytokinin responses in the CZ is a novel feature of SAM organization, whose existence was clearly revealed by the distinct absence of pTCSn1:mGFP5-ER expression in response to exogenous cytokinin . Although it has been shown that this tissue lacks significant expression of hormone-response genes, the centripetal spread of pTCSn1:mGFP5- ER expression following pCLV3:GR-LhG4 x p6xOP: ARR1ΔDDK-GR induction indicates that the absence of a cytokinin response is also accompanied by a repressive mechanism as well. Presumably this mechanism works on a protein level, as the ARR1ΔDDK-GR protein is unaffected by cytokinin signaling pathways. Given the appearance of cytokinin activity in the peripheral zone of pCLV3:GR-LhG4 x p6xOP: ARR1ΔDDK-GR plants following just 6 hours of dexamethasone treatment, this also argues against a radial degradation gradient, because the ARR1ΔDDK-GR hybrid protein would likely be destroyed before it ever reached the PZ, and degradation alone would likely still allow faint pTCSn1:mGFP5-ER activity in the CZ. Instead, it seems more likely that the ARR1ΔDDK-GR simply can’t bind to its DNA target sites, perhaps due to chromatin silencing, which might also explain the lack of hormone signalling pathway expression in these cells. However, a degradation model is consistent with the linear pWUS:eGFP-WUS concentration gradients observed in the L1-L3 tissues, whose slopes were rigidly maintained despite fluctuations in total protein concentrations . While stem cell identity is known to require the migration of WUS proteins into the overlying CZ cells, the response-free zone continues to be clearly visible even in wus-1 mutants. The zone was also present in clv3-2 mutants, and the lack of response by pCLV3:mGFP5-ER to a variety of different cytokinin treatments strongly suggests that CLV3 expression is not regulated by cytokinin responses. One possible exception is the reduced expression levels of CLV3 in ahk2/3/4 plants, though the near-absence of WUS proteins in this mutant background might suggest that CLV3 is simply not strongly activated. The potential link between cytokinin and WUS transcription is a bit harder to dis-entangle though, as ectopic cytokinin responses produced by the pCLV3:GR-LhG4 x p6xOP: ARR1ΔDDK-GR system did significantly increase the number of WUS-expressing cells, all ofwhich also had strong cytokinin responses by 48 hours. Close examination of RNA in-situ images however, reveals that WUS is expressed even in the complete absence of cytokinin responses, both in subsets of SAM tissue in the two-component system, and in the ahk2/3/4 mutant. However, this pattern may be tissue-specific, as the RNA in-situ images also show that WUS is not strongly expressed in the L1 and L2 of the pCLV3:GR-LhG4 x p6xOP: ARR1ΔDDK-GR system, drainage pot which is true whether or not cytokinin responses occur in those cells. In addition, the pWUS-eGFP-WUS fluorescence level was also largely unchanged when cytokinin levels were reduced with the p35S:GR-LhG4::p6xOP:CKX3 construct. Based on these observations, it seems quite likely that WUS transcription responds directly to cytokinin responses. Although the number of WUS-expressing cells does dramatically increase following prolonged induction of cytokinin responses in the response-free zone, this appears to be a secondary effect that occurs after the cells have acquired stem cell identity.In the conditions used by the present study, elimination of the cytokinin-response free zone could only be achieved with the pCLV3:GR-LhG4 x p6xOP: ARR1ΔDDK-GR system. This does not rule out a negative regulatory pathway though, as weak pTCSn1:mGFP5-ER expression patterns were found in the L1 and L2 cells of clv3-2 mutants . In addition, the weak expression pattern also produced a gradient from L3 up to L1 cells, which is consistent with non-cell autonomous movement of cytokinin response proteins.

Although the present data cannot identify which proteins might be involved, the most likely candidates would be members of the cytokinin signal transduction pathway, including Arabidopsis Histidine Phosphatase and ARR family proteins. However, in most cases the movements of these native proteins have not yet been studied. The sole exception is ARR7, which when ectopically expressed in L1 cells, was found to move by at least one cell layer. Presumably, if exogenous cytokinin were applied at high enough concentrations, such non-cell autonomous movement might be sufficient to repress the response-free zone even in WT plants, eventually inducing cell proliferation and WUS expression. Although this experiment was not attempted by the present study, it is interesting to note when extremely high cytokinin concentrations have been used, the SAM has been shown to have higher WUS transcript levels. Exogenous cytokinin applications have even been found to produce a downward expansion of the WUS-expressing cell volume, similar to the results of the pCLV3:GRLhG4 x p6xOP: ARR1ΔDDK-GR system in the present study. Thus it would thus be of interest to determine if the cytokinin-induced increase in WUS transcript levels is due to an increase in the number of cells expressing WUS, or if this reflects an increase in the amount of WUS transcripts per individual cell.From a developmental standpoint, the cytokinin-response free zone appears to be required in order suppress cell division in the underlying RM. This is supported by the massive cell proliferation observed following the loss of the response-free zone in induced pCLV3:GR-LhG4 x ARR1ΔDDK-GR plants. While it is tempting to speculate that the response-free zone is needed to produce a downwardly mobile morphogen that stimulates such proliferation, the elimination of the source would likely produce shoot-ward patterns as the morphogen concentration gradient decays, rather than the rootward pattern that is actually observed. Instead, the suppression of both WUS and CLV3 expression around lateral anlagen even after prolonged dexamethasone treatment in the pCLV3:GR-LhG4 x ARR1ΔDDK-GR background suggests that the repressive signalactually originates in the PZ. As the CZ is known to produce auxin biosynthesis genes CZ, and that cytokinin has repeatedly been found to reduce auxin transport , a likely model suggests that the ectopic cytokinin response in the CZ blocks auxin transport to the PZ. The subsequent failure to activate repressive auxin response factors in the PZ might then favor proliferation over cell elongation.In a developmental context, nuclear trapping has repeatedly been shown to restrict the movement of transcription factors to a single cell layer. The extended range of WUS protein movement over 3-5 cell layers is somewhat inconsistent with a full nuclear trapping model, though the pWUS:eGFP-WUS reporter does clearly show a moderate nuclear pattern. However, the nuclear localization of WUS was found to be largely independent of cytokinin responses, though two other patterns were found instead. The first of these was the enlarged nuclear volume, which was clearly cytokinin-dependent. Similar enlarged nuclei in other angiosperms have been correlated with endo-reduplication, and this is consistent with the enhanced cell proliferation rates seen under prolonged chemical treatments. The absence of any change in the WUS nuclear/cytoplasmic ratio is most easily explained a passive process, as dilution of WUS in an enlarged nucleus would be precisely balanced by an increase in WUS concentration in the cytoplasm, so long as the total cell volume itself did not change. The failure of protein re-distribution to occur following the nuclear volume is harder to explain, as active transport mechanisms through the nuclear pore should presumably restore the original concentrations within a few minutes. No such equilibrium adjustment was detected in the present study, which counter-intuitively suggests that WUS only has a limited ability to move through the nuclear pore. This may reflect the mass of the eGFP-WUS protein, which at 64kDa, is much larger than the 40kDa passive diffusion limit of the nuclear pore.

The steady decline in the distance between meristems as previously observed , may simply reflect the decreasing size of the central area. Such an effect may be widespread among angiosperms, as bud rates increased following induction in chrysanthemums and Chenopodium amaranticolor, Protea spp., while increased meristem sizes following induction have been found in apple, strawberry, chrysanthemums, and Protea spp.. At about the time the first flower reaches anthesis, the inflorescence meristem then begins a long steady decline in activity. This begins well before the rosette leaves begin to senesce, andcontinues unperturbed well after most leaves are gone . A similar declining pattern was also found in continually de-fruited plants . Both of these patterns are poorly correlated with the supply of carbohydrates, which suggests that this growth pattern is actually the product of a different developmental program. One clue about the nature of the other developmental mechanism might come from an age-related response of the meristem maintenance gene WUSCHEL, which was reduced over time in the clv3-2 background. This may reflect a broader phenomenon, as the IM tissues compared in the present study were of considerably different ages. The failure to detect more than 8000 genes in the present study could thus be a result of an age-related decline in transcriptional activity, rather than the result of fruitload effects. Additional evidence of a non-carbohydrate limited developmental mechanism comes from the analysis of de-fruited plants, which displayed a peculiar pattern of prolonged activity that began at almost just before WT plants began to reach meristem arrest . This also corresponds to a time when the majority of leaves have already senesced, plant pot with drainage implying that the trigger for meristem arrest is at least partially independent of carbohydrate supply.

This can also be seen in the soc1/ful double mutant, which maintains large numbers of leaves throughout its lifespan, and thus has an abundant carbohydrate supply. However, under continuous light, it was found that the soc1/ful IM began to break down after producing approximately 60 lateral organs , a figure that is remarkably similar to the maximum number obtained with de-fruited Col-0 plants , and with male sterile plants in the Ler background. Thus it would appear that maximum lifespan of an IM is genetically determined, though carbohydrate supplies probably trigger arrest at a predictable point long before the maximum is reached. However, it remains unclear why that predictable point occurs at approximately 40 flowers, as leaf senescence patterns would favor an earlier time point.Interestingly, calculations based on the diameter of the Arabidopsis meristem at induction combined with the reported 10% decrease between spacing of successive flower primorida and the minimum size of meristem cells suggest that the IM is capable of producing about 20 flowers before the size of the meristem drops below a single cell diameter. Although this is only half of the actual number of buds that are produced on most plants, this estimate is a close match for the number of flowers produced after the start of the declining trend, 15 days after induction. This suggests that meristem maintenance pathways may be turned off as early as the first open flower, while subsequent flower bud production merely consumes the remaining stem cells.Once an inflorescence meristem has reached a quiescent phase, it will eventually be permanently inactivated by one of two different mechanisms. In the Ler ecotype, it was reported that certain meristems eventually produced a number of carpel-like bracts before being entirely lost.

A similar pattern is observed in the soc1/ful mutant, which often produces carpel-like bracts over an extended period of time, and even rosette-like whorls of small leaves in place of the flowers. These anomalous carpel-like structures have also been reported for other Brassica spp., suggesting the existence of a single mechanism derived from a common ancestor. In this case however, even carbohydrate starvation can be ruled out, as soc1/ful double mutants retain large numbers of green leaves throughout the time of growth arrest . The mixing of different developmental programs in these terminal structures further implies that the meristem gradually loses its ability to define organ identities. This is perhaps not unique to Arabidopsis, as it has also been observed in vegetative apices of a broad array of angiosperm species, including blueberries, elms, willows, mulberry, peas, kiwifruit, lilac, Brownea ariza , maple trees, Tilia chordata, Theobroma cacao and alternate bearing pistachios. The localized nature of the senescent pattern suggests that only a small group of cells actually perceive the triggering event. Combined with the observation that multiple nearby tissues senescence simultaneously, even when they are not in direct contact with each other, this also strongly suggests the involvement of the ethylene hormone. The most likely hypothesis to explain this pattern is an ethylene “burst”, similar to the pattern of ethylene production that occurs in climacteric fruit. Provided ethylene biosynthesis was of short duration, this would be sufficient to explain the simultaneous senescence of the meristem and adjacent flower buds and the limited range of the senescent signal.

Intriguingly, the ethylene biosynthesis gene ACS5 does appear to be upregulated in the apices of old inflorescence meristems, which correlates well the up-regulation of copper-related genes found in the present study, which may result in enhanced ethylene perception. The weakening of the cell wall predicted suggested by xyloglucan hydrolase TCH4, the starch degradation and even the expression of PAP2 to produce red pigments in the present study also closely parallel the process of fruit ripening in other species, which involve an increase in pectinesterases, an increase in free sugars and the synthesis of anthocyanin pigments.To estimate meristem activity in terms of the rate of new anlagen production, Col-0 plants were grown in long-day conditions . The number of immature flower buds between about stage 4 and stage 11 were counted every other day on the 1° meristem, along with the number of mature flowers and fruits. Stem height was measured by placing one end of a 2.5 cm wide ruler on top of the rosette as the stem began to bolt, recording the position of the flat apical region, not the highest flower bud. The flower buds were visualized with 10x magnification, allowing minimally invasive and repeated counts of the same plant.Leaf lengths were measured every two days until they began to senesce. This was then used to plot the average growth rates, identifying their length at the time of their maximum growth rate. Growth rates during their first six days were accurately predicted by the empirically determined formula: Leaf length = 1.05t2 , where length is in millimeters and “t” is measured in days. This was used to calculate the age of the leaf at the time of its first measureable length, then subtracted from the chronological plant age to obtain the time of leaf initiation. Leaf tip removal was timed to occur just after maximum leaf growth, on a leaf by leaf basis. Roughly 1/3 of the total leaf area was removed, using two cuts to remove a diamond-shaped section, reflecting the pattern of senescence. Stem heights, buds and fruits numbers were measured every other day until meristem arrest became visible.Meristem collection for microarray analysis used Col-0 plants grown in both long day conditions and in continuous light. In order to maximize fruit load, but to avoid collecting senescent tissues, pots with drainage holes all plants were harvested simultaneously when a single meristem anywhere in the flat was found to be arrested, under the expectation that the rest would become senescent within 48 hours as previously established. Meristem tissue collection proceeded in a stepwise manner, first by harvesting 3-5 cm sections of Arabidopsis branch tips, followed by micro-dissection of the SAM in batches of 10-15 meristems. These included all 1°, 2°, 3° and higher order branch meristems, as available. All flowers and fruits older than stage 2-3 were surgically removed from each section using a dissecting microscope, first removing mature flower buds and fruits, then by collecting a volume of SAM tissue of equal height and width, measuring roughly 0.2mm3 . The same blade and cutting surface were used for all cuts. The SAM tissue was flash-frozen in liquid N2 within 30 seconds of dissection. A total of about 1500 meristems were pooled for each biological replicate, for a total of five replicates. RNA was extracted from the plant tissue by grinding the tissue under liquid nitrogen with Triazol reagent, then extracted with a Qaigen RNA easy kit.The ability of plants to grow from seemingly nothing at all has fascinated people for millenia, and was perhaps most famously demonstrated by Johannes Baptista van Helmont’s 1648 potted willow experiment. Although he incorrectly deduced that plants obtained their mass from water, it is known today that plants obtain much of their bulk from carbon dioxide in the atmosphere.

This gas is then used by the plant to make sugars, cellulose and other molecules needed to grow and reproduce. However, it is also equally clear that plants do not simply swell up like a sponge, because their seedlings bear little resemblance to mature trees. Instead, plants actually confine much of their growth and development to very small parts of their anatomy, which can be found by tracing the stem from the ground up, so to speak. After passing the trunk, the larger branches, and the slender twigs, the actual tip of the branch is often found to be obscured by a dense cluster of small scales or leaves, collectively known as a bud. When these leaves are peeled off layer-bylayer, one will eventually find a smooth rounded dome in the center, often a mere fraction of a millimeter wide, surrounded by tiny organs in various stages of development. This is the primary unit of plant growth, and it is known as the shoot apical meristem . Appearances can be deceiving however, as the SAM actually performs many critically important activities necessary for survival. The more obvious of these include the production of all new leaves, branches, and flowers, which replace lost or damaged organs, and are necessary to produce the next generation. The SAM is also the site of many developmentally important decisions, regulating aspects such as how fast the plant grows, how many leaves are produced, and when to flower. These decisions are in turn are based on a wide variety of information sources, such as temperature, photoperiod, disease, age, and the current nutritional state of the plant.Unlike an animal brain though, very little of this decision-making process is evident in the cellular anatomy of the SAM. When examined from longitudinal sections, its tissues are slightly smaller and denser than average, but there is otherwise little to attract attention. The most obvious feature is a subtle layered arrangement of cells near the surface, which are distinguishable by the fact that their cells always divide at right angles to the surface. In most flowering plants, two such layers are present, each of which is a single cell thick, and both are draped over an interal dome-shaped mound of irregularly shaped cells. For the sake of convenience, the layers are numbered from the outside-in starting with Layer 1 , and then proceeding through L2, L3, and so on. However, this system becomes less useful with tissue depth, because the presence of the irregularly shaped cells deep inside the SAM make it increasingly difficult to identify the individual layers. As a result, many authors simply stop counting at L3, but it is commonly accepted that “L3” refers to the entire inner volume of irregularly shaped cells, rather than to a single layer . The three-layered description has some support in terms of known gene expression patterns, and to avoid confusion the remainder of this dissertation will also stop counting at L3. The only exception occurs in chapter 4, where a longitudinal analysis of protein distributions made it necessary to describe cell layers as deep as L11 . In addition to the cell layers, there is also another discrete set of patterns in the SAM that cannot be seen by the naked eye. They are instead recognized by differences in gene expression patterns and cell division rates. The very center of the SAM for example, contains a vertical column of cells that divide at rates 2-3x slower than those in the periphery.

Eight of these genes were strongly upregulated in all four treatments, indicating that B. cinerea may produce these toxins regardless of the ripening stage of the fruit. F. acuminatum genes annotated with this GO term included enzymes involved in the biosynthesis of the toxin fumonisin. Several of these genes showed significant upregulation in infections of MG fruit at 1 dpi or RR fruit at both time points. Fungal proteolysis-related genes were found to be enriched during MG and RR inoculations with F. acuminatum as well as RR inoculations with R. stolonifer at 3 dpi. Though not enriched, several genes with this GO term were also found to be expressed during fruit inoculation by B. cinerea, mostly in RR fruit. Across all treatments, F. acuminatum was found to produce 28 genes with this GO term, while B. cinerea was found to produce 29, and R. stolonifer produced 44 in RR fruit alone . Seven members of the B. cinerea aspartic proteinase family were upregulated in at least one of the fruit inoculations, though none were upregulated at 1 dpi in RR fruit. Thus, fungal proteases are likely to be a strategy used by all three pathogens. Other GO terms served as a proxy for successful growth. Enrichments of genes involved in protein translation initiation , glycolytic process , and DNA replication initiation were found in compatible interactions with RR fruit. Notably, DEGs involved in glycolytic process were enriched in MG inoculations for F. acuminatum at 1 dpi, which is consistent with visual observations of mycelium growth on inoculated fruit. A similar pattern was observed for the chitin catabolic process term, vertical aeroponic tower garden which are involved in the continuous fungal cell wall remodeling during hyphal growth . Multiple GO terms relating to carbohydrate metabolism were found to be enriched across multiple fruit inoculation treatments.

The corresponding genes included those involved in breakdown of the cell wall polysaccharides, metabolism of host sugar sources, and production of fungal polysaccharides. As both the cell wall properties and sugar biochemistry differ between MG and RR fruit, we hypothesized that the fungi employ different classes of CAZymes depending on the ripening stage as already demonstrated for B. cinerea . To test this, we examined the expression profiles of CAZyme families among the DEGs for each pathogen . CAZyme families involved in catabolism of cellulose, hemicellulose, pectin, and monosaccharides were detected, along with families with non-carbohydrate substrates and several responsible for polysaccharide biosynthesis. In B. cinerea and F. acuminatum inoculations, families involved in the degradation of cellulose and hemicellulose were more prominent during infections of MG fruit than RR fruit. Moreover, the CE5 family, which contains cutinases and acetylxylan esterases, was also especially utilized at 1 dpi in MG fruit. In B. cinerea, this family included the cutA gene previously shown to be expressed in tomato fruit infection . MG infections also exhibited higher percentages of families involved in the degradation of cellobiose, a disaccharide of β-1,4-linked glucose molecules that results from the breakdown of cellulose and glucan-based hemicelluloses. A similar trend was found for pectin-degrading families, particularly polygalacturonases and pectate lyases , though the PL1-4 subfamily appeared to be prominent in RR infections as well. Enzymes involved in metabolism of simple sugars, most notably GH32 in F. acuminatum and AA3- 2 in B. cinerea, showed greater prominence in RR infections. Chitin and chitosan biosynthesis and processing families were also detected in B. cinerea and F. acuminatum. In B. cinerea, chitin synthases were generally equally expressed in all fruit inoculations, though chitin deacetylases , which produce chitosan, were only particularly prominent in RR infections at 3 dpi.

In contrast, F. acuminatum produces multiple CE4 enzymes at 3 dpi in MG infections in addition to RR infections. CE10 enzymes were especially prevalent in both B. cinerea and F. acuminatum infections. Members of the CE10 family include lipases, which catalyze the hydrolysis of fatty acids. The previously described B. cinerea gene lip1 was upregulated at both 1 and 3 dpi in MG fruit, but not RR fruit. Additionally, B. cinerea and F. acuminatum both produced multiple AA7 family enzymes in both MG and RR fruit. Many of these genes showed significant similarity to three genes of the PHI database: ZEB1 in F. graminearum, CTB5 from Cercospora nicotianae, and sol5 from Alternaria solani. Each of these PHI genes is involved in the biosynthesis of polyketide mycotoxins in those plant pathogens . Thus, these B. cinerea and F. acuminatum genes may be involved in similar roles. Detection of CAZymes during infection by R. stolonifer was only possible in RR fruit due to the low number of DEGs determined in MG fruit. However, sizable numbers of genes from families detected in B. cinerea and F. acuminatum infections were also discovered in R. stolonifer. These include xyloglucanases , cellobiose dehydrogenases , pectin methylesterases , and polygalacturonases . In addition, multiple enzymes involved in chitin/chitosan biosynthesis were prevalent in RR fruit inoculations, which is indicative of the particularly aggressive hyphal growth of R. stolonifer on these fruit. In RR fruit at 3 dpi, R. stolonifer also produced six enzymes of the AA1 family, which consist of laccases, ferroxidases, and multi-copper oxidases. Each of these enzymes showed significant similarity to FET3 enzymes from Colletotrichum graminicola in the PHI database and to genes of the TCDB class 2.A.108.1.4, the latter being iron transport multicopper oxidase FET5 precursors.

This finding is also consistent with the enrichment of transmembrane transport genes during RR infection at 3 dpi for R. stolonifer.We inoculated fruit of the non-ripening tomato mutant to verify the effect of the ripening stage on the infection success of B. cinerea, F. acuminatum and R. stolonifer. Fruit from the nor mutant do not show ripening-associated processes, such as carotenoid and sugar accumulation or cell wall disassembly, and therefore resemble wild-type MG fruit even at a comparative RR-like stage. None of the three pathogens were able to infect nor fruit at any ripening stage . No hyphal growth of B. cinerea and R. stolonifer was apparent, whereas F. acuminatum formed visible mycelia especially at 3 dpi in MG and RR-like fruit. Like on wild-type MG fruit, all three fungi induced necrotic rings in nor fruit. When inoculated in RR-like fruit from nor, the three fungi displayed similar growth and morphology as in MG fruit from wild-type and nor, indicating that for compatible interactions to occur, tomato fruit needs to undergo certain ripening processes that facilitate fungal colonization and spread.Successful infections of B. cinerea, F. acuminatum and R. stolonifer in tomato fruit are dependent on the host developmental stage. In MG fruit, all three fungi were able to grow on the surface, but none of the pathogens was able to cause rot. In contrast, ripe tomato fruit represented a compatible system for infections as all three fungi induced lesions that spread rapidly. This contrasting ability to cause disease in fruit has been previously reported for a variety of fungal pathogens, particularly those displaying necrotrophic behavior . The tomato gene SlWRKY33 has been shown in leaves to be expressed in response to B. cinerea inoculation, vertical gardening in greenhouse and deletion of this gene leads to increased susceptibility, indicating its role in defense response . We demonstrated that, as in leaves, B. cinerea is capable of inducing SlWRKY33 in MG and RR fruit. Furthermore, F. acuminatum also induced SlWRKY33 in MG and RR fruit, and R. stolonifer did so substantially in RR fruit. These findings indicate that all three pathogens triggered disease responses in the host and that the strength of the response was reflective on the success of the infection process. During interactions with tomato fruit, B. cinerea, F. acuminatum, and R. stolonifer employed a variety of pathogenicity and survival strategies that involved redox processes, carbohydrate catabolism, and proteolysis. Moreover, the degree to which particular strategies were used varied according to the ripening stage of the fruit, as certain processes were emphasized in either MG or RR fruit . These observations suggest that the fungi can sense the physiological environment of the fruit and react accordingly with suitable infection, growth, or quiescence strategies. Though these fungi are incapable of causing disease symptoms in MG tomato fruit, this study demonstrates that they do make attempts to either establish infections or create a suitable environment in fruit for fungal growth and do not merely die on the host tissues. However, when the conditions in fruit are highly unsuitable , the infection strategy of the fungal pathogen is often insufficient to cause successful infections. In many cases, when fungal pathogens encounter incompatible conditions, like in unripe fruit, they enter a quiescent phase with limited growth and activity . During ripening, the physicochemical properties of the fruit tissues change, resulting in compatible conditions for the fruit-pathogen interaction and the reactivation of quiescent pathogens . In this context, it would also be interesting to investigate the strategies employed by the three fungi during inoculations of other plant organs such as leaves.

Our initial tests, however, indicated that both F. acuminatum and R. stolonifer are incapable of infecting tomato leaves even when leaves were senescing. This observation may suggest that the isolates of these two fungi are exclusive fruit pathogens and lack the molecular toolset to grow on leaves. The redox environment of the plant-pathogen interface influences the outcome of the interaction. Upon pathogen detection, ROS are rapidly produced by the host, triggering a downstream signaling of various defense responses . The enzymatic agents of this oxidative burst are respiratory burst oxidative homologs , which generate superoxide O − 2 in the apoplast . This oxidative burst has been previously reported in incompatible tomato-Botrytis interactions , including MG fruit, in which the appearance of a necrotic ring is associated with resistance to B. cinerea . However, necrotrophic pathogens can exploit this ROS response by overwhelming the host with their own ROS production . In leaves of French bean , B. cinerea has been shown to produce ROS as virulence factors by activating the NADPH oxidases BcnoxA and BcnoxB , coupled with the regulatory protein BcnoxR . Although we did not detect strong upregulation of these genes during inoculation of fruit, other ROS producing systems, including laccases and glucose oxidases were upregulated during inoculations of tomato fruit. In F. acuminatum, a BcnoxA homolog FacuDN4838c0g1i1 and BcnoxB homolog FacuDN3221c0g1i1 were induced in specific treatments. A BLAST search did not reveal anyhomologs of BcnoxA or BcnoxB in R. stolonifer, nor were any homologs of Bclcc8 or BcGOD1 detected in either F. acuminatum or R. stolonifer. In addition to ROS generation machinery, fungal pathogens must protect themselves against the oxidative stress of the infection site. Methods of ROS scavenging in phytopathogenic fungi include enzymatic and non-enzymatic mechanisms . SODs catalyze the conversion of O− 2 produced by RBOHs into the less reactive hydrogen peroxide . B. cinerea mutants lacking the BcSOD1 gene have been shown to have reduced virulence on tomato leaves . In tomato fruit, BcSOD1 is upregulated for both MG and RR ripening stages, which suggests it is also a critical gene for fruit colonization. H2O2 can be converted to water by either catalases or peroxidases such as GPXs or PRXs. All three pathogens demonstrated upregulation of specific mechanisms of catabolizing H2O2, but only F. acuminatum showed enrichment of genes involved in the H2O2 catabolic process. The usage of these H2O2 catabolizing systems varied between the pathogens. While B. cinerea utilized catalases in MG fruit at 1 dpi, F. acuminatum and R. stolonifer produced more catalases and peroxidases in RR fruit at 1 dpi. In each pathogen, multiple genes involved in protein degradation were found to be upregulated during fruit inoculations. The strong enrichment of proteolysis-related genes may indicate that protein degradation is important for pathogenicity of F. acuminatum and R. stolonifer but not B. cinerea. Some pathogen-derived proteases, such as Sep1 and Mep1 in Fusarium oxysporum, are known to serve as suppressors of host-immune response in plant-pathogen interactions . Even though their specific roles in pathogenesis are not fully characterized, several aspartic proteinases in B. cinerea have been described . Three of the B. cinerea aspartic proteinases that we found to be induced in tomato fruit were also found to be upregulated during infection of grape berries . Aspartic proteinases were also found to be among the upregulated proteinases in F. acuminatum and R. stolonifer , though all three pathogens appeared to utilize a diverse suite of proteinases of different families.

To determine if we could identify changes in euFUL sequences or selection that might shed light on this change in function, we analyzed euFUL gene evolution in Solanaceae. We performed a maximum likelihood phylogenetic analysis  on a data set that consisted of 106 Solanaceae members of the euFUL gene lineage , which we obtained through amplification and sequencing , generating transcriptome sequence data , or mining databases . As outgroup we used 10 euFUL genes from Convolvulaceae, the sister family to Solanaceae . The resulting tree shows the two major clades of core-eudicot euFUL genes, the euFULI and euFULII lineages . Within each of these clades there is evidence of a Solanaceae-specific duplication, resulting in two subclades in each lineage. Within each subclade, the order of branches correlates well with the topology of the Solanaceae phylogeny ; discrepancies at the genus level are likely due to the short length of some sequences and sequence divergence in some taxa. Each of the subclades includes orthologs from both fleshy- and dry-fruited species, indicating that the subclade duplications preceded the origin of fleshy fruit. Although duplications in these genes are common , we did not find significant evidence of taxon-specific duplications. We did, however, find two genes that did not fall into a specific subclade. A third Streptosolen gene grouped sister to the rest of the euFULI clade , potentially the result of a taxonspecific duplication followed by sequence divergence. In addition, a Schizanthus gene grouped sister to the euFULII clade . This may also be a divergent genus-specific paralog, but since Schizanthus is one of the earliest diverging genera , square plastic pot it is also possible this gene might be a remaining paralog from the reported whole genome duplication/triplication that occurred early in Solanaceae diversification .

We also found potential evidence of loss – not every Solanaceae species we studied had a copy of each euFUL gene. We did not, for example, find FUL2 genes in Iochroma, Fabiana, Solandra, Juanulloa, Schizanthus, or Goetzia, even though these all had genes in the FUL1 clade . However, although this may represent paralog loss, it is possible we did not recover all gene copies due to PCR primer mismatches, low expression levels, or the absence of transcript in the sampled tissue. In addition to the major shift to fleshy fruit in the Solanoideae subfamily, fleshy fruits have independently evolved in Cestrum and Duboisia, and there has also been a reversal to a dry fruit in Datura . Our analysis does not include genes from Duboisia, but the euFUL genes from Cestrum and Datura grouped in positions in the tree that were expected based on their phylogenetic position, and did not show any notable differences in sequence from the euFUL genes of their close relatives.We compared dN/dS ratios between and among Solanaceae euFULI and euFULII lineages, as well as between sequences before and after the transition to fleshy fruit, to investigate if any changes in selection might be correlated with sequence diversification. All ω values from our analyses are closer to 0 than to 1 , which indicates that all euFUL gene clades are under strong purifying selection . Studies suggest that this is the norm for most protein coding genes, and that under such stringent evolutionary constraints, slight differences in evolutionary rates may result in functional diversification . Our data show a weakening of purifying selection in FUL1 genes relative to FUL2 genes and in MBP10 genes relative to MBP20 genes . Immediately after the euFULI duplication, the FUL1 and FUL2 lineage genes would have been fully redundant, which might have allowed the reduction in purifying selection on the FUL1 genes resulting in potential functional divergence.

Similarly, the duplication that resulted in the two euFULII gene clades would have resulted in redundancy in the MBP10 and MBP20 lineages, possibly allowing the more rapid diversification of MBP10 genes. Although studies indicate that the euFULI genes of tomato have novel functions compared to those in dry fruit , it remains unclear whether the new functions are the result of changes in coding sequences, regulatory regions, or downstream gene targets. Our analysis shows that euFUL genes in both dry- and fleshy-fruited species are evolving at similar rates . This suggests conservation of the coding sequences in both fleshy- and dry fruited species despite the central roles in the development of these distinct fruit morphologies. Sixty-four of the sequences in our analysis were from fleshy fruited species whereas only 42 were from dry-fruited species. Although, we had broad representation across the dry grade, it is possible with additional representation from dry fruited species, more evolutionary patterns would be revealed .An analysis of selection across an entire sequence may indicate different types of selection for the whole gene, but this overlooks the fact that key residues may be undergoing rapid evolution that may result in functional changes . Other empirical studies have further described functional changes due to a change in a single amino acid residue specifically associated with changes in polarity or conformation . Studies in A. thaliana, show that a single amino acid mutation in GLABRA1 results in the inhibition of trichome formation and a change of a single residue is sufficient to convert the function of TERMINAL FLOWER 1 , which inhibits flower formation, to that of the closely related FLOWERING LOCUS T , which promotes flowering . Three-dimensional modeling has also shown that a single amino acid change in a highly conserved domains may lead to changes in protein–protein interactions .

We searched for individual sites in the predicted amino acid sequences that showed evidence of positive selection within the gene groups that, although under purifying selection, were found to have statistically significantly accelerated evolutionary rates to determine if any amino acid changes at these sites had the potential to result in a change in protein function. Our findings show that more residues are rapidly changing in the K domain compared to the M and I domains . The K domain is predicted to have an α-helix structure that facilitates protein–protein interactions . The α-helix structure depends on conserved hydrophobic residues spaced through the domain . Therefore,changes to protein residues that alter charge and/or conformation in this region can lead to changes in such interactions. Most of the rapidly evolving sites did not show an amino acid change specifically associated with the shift to fleshy fruit, but rather showed changes and reversals over the course of gene evolution. Interestingly, in the FUL1 proteins, we found one site in the K domain, corresponding to the 153rd residue in the tomato protein , at which 11 out of 15 sequences from dry-fruited species have a negatively charged glutamate residue. In comparison, 100% of the fleshy clade contains a nonpolar residue: valine  or methionine . However, since the remaining four FUL1 sequences from dryfruited species have non-polar glutamine or V at this site, the change from charged to non-polar is not associated with the shift to fleshy fruit. In addition, a PROVEAN analysis predicted the changes at this site to be neutral with regards to function. Two other sites in the FUL1 K domain show changes that are predicted to have functionally deleterious consequences according to our PROVEAN analysis . These include a charged histidine to a non-polar glutamine/asparagine transition at the 95th residue and a charged lysine to non-polar glutamine/threonine transition at the 157th residue . Polar residues are important for protein–protein interactions of the K domain α-helix and changes might disrupt interactions with other proteins . However, since these changes are not correlated with the fruit type, it seems unlikely that any alteration to protein function affects fruit morphology. It is also plausible that any negative effect at these sites is masked by the FUL2 paralog, which is likely to be functionally redundant . This is consistent with FUL1 evolving relatively faster , thus enabling divergence compared to FUL2, 25 liter pot which appears to be more highly functionally conserved based on stricter sequence conservation. None of the sites undergoing positive change in the K domain of MBP10 showed a change in charge, suggesting these changes are not likely to affect protein function. We also observed residues in the M domain that are under diversifying selection in both the FUL1 and MBP10 clades. These residues are located not in the α-helix region that directly binds to DNA, but in the β-sheet region of the MADS domain . β-sheets are important for protein arrangement in three dimensional space. Therefore, any changes in this region might change protein conformation, influencing DNA binding of the α-helix as well as the ability of the euFUL proteins to form higher order complexes . However, these shifts were reversible, with no phylogenetic pattern or change in charge, and there was no correlation with the fruit type. Therefore it is unlikely that these shifts have significant functional impact. A previous report that investigated the evolution of MADSbox genes in A. thaliana also found rapidly evolving sites in the M and K domains of Type II MADS-box proteins, which might have been involved in the functional diversification of this group, but did not report changes in the I domain .

Residues in this domain that are directly involved in forming an α-helix structure are expected to be highly conserved, whereas the remaining residues may not be under such constraints . We found residues in the conserved region of the I domain that are undergoing diversifying selection in both FUL1 and MBP10 clades. Of these, one site in FUL1 and three sites in MBP10 had undergone changes in charge but none were predicted to negatively affect the function . In addition, as with the sites in the M and K domains, none of these was correlated with the Solanaceae phylogeny or changes in fruit morphology. It has been reported that higher rates of substitution in lineages that show weakened purifying selection or even diversifying selection may be occurring at residues of minimal functional importance . This might explain the apparent ease of reversibility and lack of phylogenetic signal among the rapidly changing sites we observed.independent of the reported whole genome events, occurring prior to the diversification of the Brunfelsia clade but after the event that produced the FUL1 and FUL2 clades . The expected topology for the euFULII clade, based on a duplication prior to the divergence of the Brunfelsia clade, would be a paraphyletic grade of pre-duplication euFULII genes, from species that diversified prior to Brunfelsia, and nested MBP10 and MBP20 clades that would include post-duplication genes from all species that diversified subsequent to the duplication. However, in our tree, the pre-duplication genes do not form such a basal grade . Rather, they form a clade with the post-duplication MBP20 genes. The results of our PAML analyses indicate that the MBP20-clade genes show less sequence divergence than MBP10 genes; this higher degree of similarity among pre-duplication sequences and post-duplication MBP20 genes may underlie their grouping into one clade . Our results indicate that the euFULII duplication occurred prior to the origin of the clade containing Brunfelsia. We would therefore expect to find both an MBP10 and an MBP20 in all species of that clade. However, we did not find an MBP10 ortholog in members of this clade other than Brunfelsia. MBP10 appears to have been lost from the genome of Petunia, based on analyses of multiple fully sequenced genomes , and potentially from Plowmania and Fabiana. We were able to recover MBP10 orthologs from Nicotiana and most other later-diverging genera. However, our analysis includes fewer species from the dry grade of the Solanaceae phylogeny than the fleshy-fruited Solanoideae clade and even fewer species that diverged prior to Brunfelsia . In the MBP10 clade in particular, our analysis includes 13 orthologs from species in the fleshy-fruited clade but just four from the dryfruited species, and our analysis only includes sequence data from four genera that diverged prior to the origin of the Brunfelsia clade . Thus there may be genera that originated prior to Brunfelsia that contain MBP10 that our sampling did not include. Floral and fruit transcriptomes, which provided MBP10 orthologs from later diverging species, yielded no MBP10 sequences from Cestrum and Schizanthus; nonetheless, whole genome sequences of early diverging species are needed to determine the timing of the MBP10/MBP20 duplication.

The irrigation frequency was increased to 5 times per day with 200 mL at full bloom . It was then increased to 8 times per day the week after pollination . Irrigation started 1 h before sunrise and finished 1 h after sunset. Three days before harvest, the irrigation frequency was adjusted to one time per day with 4800 mL water to further enhance cracking. Plants were topped on 28 February when they had 2 clusters of flowers and were pollinated on 1, 4, 8, and 11 March. On 18 March, the spray treatments with water or ABA began, applied 3 times per week for 7 weeks, until 22 April. Tomato cracking rate, firmness, total soluble solids and titratable acidity were analyzed on 30 April. The fruit materials for other analyses were preserved until the next day and then analyzed.Previous research indicates that heritable resistance to cracking can be identified in some tomato breeding lines. However, no single genetic locus seems to be responsible for inheritance of the fruit cracking trait and many genes may contribute to the phenotype. Many studies point to the involvement of cell wall structure and possibly the cuticle layer in fruit cracking. As cell wall networks weaken with fruit ripening, even as cell turgor falls, resistance to stresses at the fruit surface may require a greater contribution from wax and cuticle layer structures than they can provide; and cell turgor pushing the plasmalemma against the cell wall also creates some stress on the cell wall polysaccharide networks that may be accommodated by the elasticity of the wall “fabric”; this then leading to cracking. Polysaccharides make up more than 90% of the mass of the plant cell wall. The pectins are relatively uronic acidrich polymers that are the most structurally complex polysaccharides in plant primary cell walls.

PG is believed to be responsible for a large part of the HG pectin depolymerization in ripening tomatoes; PG mRNA, protein, dutch buckets and activity accumulate to very high levels late in the ripening of tomato fruit. Brummell reported that suppression of the ripening-related EXP-encoding gene slowed tomato fruit softening early in ripening, and theyhypothesized that EXP1-mediated relaxation of the wall structure is necessary to allow PG or other enzymes access to polyuronide or other wall substrates. To investigate how PG and EXP may work collaboratively to affect the susceptibility of tomato fruit to cracking, we investigated differences in cell wall composition as influenced by the pg/exp genotype in fruit stressed by increased water uptake following treatment with ABA. Ripening in tomato is accompanied by a shift in pectins from the CSP and SSP to the WSP. The clearest impact of simultaneous suppression of PG and EXP was that the pg/exp fruit displayed a substantially reduced breakdown of the cell wall pectin network as they proceed through ripening and the fruit soften less than WT fruit. The pectin polymers in ripe fruit of the pg/exp genotype are bigger than those in ripe WT fruit, and there was more SSP in pg/exp fruit compared with WT. In our study, the cracking-resistant pg/exp genotype, had more CSP in the mesocarp portion, and more SSP in the exocarp portion of the fruit. In contrast, there was more WSP in fruit of the WT genotype. This observation suggests that both the exocarp and mesocarp cell walls of pg/exp fruit were more intact and thus better able to resist internal stresses that are presumed to promote ripe fruit cracking. The calcium content of the fruit and their cell walls can affect cell wall strength.

Pectins with low levels of methoxyl-esterification can form gels; calcium-ion bridging of unesterified GalA residue carboxyl groups on neighboring HG pectins has been proposed to form “eggbox” structures in the primary cell wall matrix. And the strength of the Ca2+-promoted-gels increases with increasing Ca2+ concentration. In this experiment, the higher Ca2+ level in the AIR from WT fruit than in the AIR from the firmer and less-cracked pg/exp fruit is somewhat surprising. It is not clear how the AIR’s Ca2+ content corresponds with the relative distributions of Ca2+ in the cell wall/apoplast, the cytoplasm and the vacuolar compartments. However, our immuno- fluorescence microscopy with JIM5 and JIM7 antibodies revealed that pg/exp fruit had more highly esterified pectins than WT fruit, indicating less capacity for cell wall binding of Ca2+. In some cases, there is also less Ca2+ in cracking-resistant varieties. We conclude that a ripe fruit with more intact pectins in its primary walls is likely to resist cracking more effectively, as long as a reasonable degree of pectin-pectin bonding is retained. The correlation analysis demonstrated that crack rate was associated most significantly with the protopectin and cellulose rather than Ca2+, which confirms this view. In our study, we used whole-plant sprays of ABA to increase the tendency of tomato fruit to crack. ABA application can decrease stomatal conductance and leaf transpiration, and increases plant water potential, which results in significantly increased xylemic flow into tomato fruit. This xylemic flow also carries more Ca2+ into the fruit as has been reported previously and is evident in the higher Ca2+ levels in both whole fruit and cell walls of ABA treated fruit.

The higher incidence of cracking in ABA treated RR tomato fruit was likely due to accumulation of water in the fruit when leaf transpiration was reduced by ABA, likely resulting in increases in turgor pressure in the fruit. However, the fruit genotypes showed significant differences in their tendency to crack when cracking was promoted by ABA application. The increase in cracking in response to ABA treatment was not observed in RR pg/exp fruit but was observed in WT and glk2 fruit. And there was no difference in cracking among the three genotypes when plants were treated with water. ABA treatment also had an influence on tomato cell wall composition, resulting in lower amounts of WSP and SSP in mesocarp and blossom end tissues of all three genotypes. This does not appear to be related to influences on fruit ripening as no visible differences in ripening were observed and ABA is generally reported to enhance ripening, not slow ripening. The higher proportion of chelator soluble cell wall material may be a response to the higher Ca2+ levels in the fruit due to the higher xylemic flow, but it is unclear what role if any this played in the increased cracking of ABA treated fruit. Cell walls from pg/exp fruit also appeared thicker and denser than cell walls from WT fruit under electron microscopy, perhaps because of reduced disassembly of the cell wall polysaccharide polymer network. This difference could be another reason for resistance to cracking in this genotype. The thicker and denser cell walls from pg/exp fruit are reflected in the higher levels of CSP, SSP and cellulose in cell wall extracts prepared from the mesocarp of pg/exp fruit. Cantu previously demonstrated that the reduction of both PG and EXP activities resulted in isolated and in situ cell walls that swelled much less than walls from WT fruit that soften significantly at the fully ripe stage; which supports the conclusion that pg/exp fruit has a more intact cell wall than WT fruit. In our experiments, pg/exp had a thicker and denser cell wall that may resist swelling. The cuticular wax layer was thicker in pg/exp fruit, which could also contribute to resistance to cracking. While waxes are part of the overall extracellular matrix, they are not targets of either PG or EXP, which suggests that suppression of the ripening-associated SlPG and SlEXP1 genes may also have impacts on other structures at the fruit surface. It is interesting to note that in addition to changing cell wall network integrity, tomato fruit cuticle chemistry, grow bucket and structure have been identified as fruit factors that influence ripening-associated fruit softening. The correlation analysis also showed that cracking rate was significantly associated with cell wall composition and cell wall-thickness, as well as with wax-thickness. Since increased water uptake by the fruit due to exposure of the plants to ABA promotes cracking, the physical constraints of the more intact cell walls in the pg/exp fruit seem to be the major factor providing resistance to cracking at the later stage of ripening .Approximately one-third of food produced globally is lost or wasted , yet fewer resources are devoted to post harvest research and development than to efforts for improving productivity. The modular design of plants allows plant tissues and organs to remain biologically active even after harvest. Therefore, capitalizing on the ability of harvested vegetables and fruits to continue to sense and respond to diverse stimuli, similarly to intact plants, may be a powerful approach to promote post harvest quality. Research demonstrating the biological advantage of a functional circadian clock in plants led us to investigate whether maintaining diurnal cycles may promote longevity and therefore reduced yield loss during post harvest storage of vegetables.

The circadian clock enables plants to anticipate and prepare for the daily environmental changes that occur as a consequence of the rotation of the earth. Coordination of plant circadian rhythms with the external environment provides growth and reproductive advantages to plants, as well as enhanced resistance to insects and pathogens. The circadian clock also regulates aspects of plant biology that may have human health impact, such as levels of carbohydrates, ascorbic acid, chlorophyll , and glucosinolates in edible plant species. Plants exhibit exquisite sensitivity to light stimuli, and isolated plant leaves maintain responsiveness to light after harvest and can continue light-dependent biological processes, such as photosynthesis. Additionally, the clocks of post harvest fruit and vegetable tissues can been trained with 12-hour light/12-hour darkness cycles producing rhythmic behaviors not observed in tissues stored in constant light or constant dark. For example, light exposure delays broccoli senescence and yellowing but accelerates browning in cauliflower, a close relative of broccoli. Other studies report that light exposure to broccoli during post harvest storage either provides no additional benefits or decreases performance. Post harvest light exposure improves chlorophyll content in cabbage, but leads to increased browning of romaine lettuce leaves. Although exposure of spinach to light during post harvest storage can improve nutritional value, light can also accelerate spinach water loss, leading to wilting. Together, these findings are inconclusive as to whether light exposure during post harvest storage can be generally beneficial, and the variation of the results may be attributable to differences in the plant species examined and the specific conditions used during post harvest storage, such as lighting intensities, temperature, humidity or packaging. Alternatively, light may be advantageous but only if present in its natural context with 24-hour periodicity because of such timing on circadian clock function. This study aimed to examine whether mimicking aspects of the natural environment predicted to maintain circadian biological rhythms during post harvest storage of green leafy vegetables improves performance and longevity compared to post harvest storage under constant light or constant darkness. We focused this work on several popular and nutritionally valuable species, including kale and cabbage , members of the Brassicaceae family with worldwide production of approximately 70 million tons. In addition, we analyzed green leaf lettuce and spinach , which have worldwide production of approximately 25 and 22 million tons, respectively. Here, we report on the promotion of post harvest longevity, including tissue integrity and nutritional value, of green leafy vegetables by provision of 24-hour light/dark cycles during storage compared to storage under constant light or constant darkness.Fruits and vegetables after harvest can respond to repeated cycles of 12-hour light/12-hour dark, resulting in circadian clock function and rhythmic behaviors. Because a functional plant circadian clock is physiologically advantageous we sought to address whether post harvest storage under conditions that simulate day/night cycles, thereby potentially maintaining biological rhythms, would affect post harvest longevity. We chose to address this question using green leafy vegetables, including commonly consumed kale , cabbage , green leaf lettuce and spinach , because we anticipated that the leaf organ would likely maintain light sensitivity and responsiveness even after harvest. To begin to determine whether daily light/dark cycles during post harvest storage affects leaf longevity, we compared the overall appearance of leaf disks that were stored at 22°C under cycles of 12-hour light/12-hour darkness versus leaf disks stored under constant light or constant darkness for various lengths of time . Under cycles of 12-hour light/12-hour darkness, kale leaf disks were dark green after 3 days of storage .

OMT3 was found to be the major genetic determinant for this trait in two independent studies. Nevertheless, it is possible that OMT1 may contribute to the IBMP concentration, because OMT1 can synthesize IBMP and it is located at the edge of a QTL significantly contributing to this trait. Furthermore, the majority of IBHP , the precursor for the OMT1 and OMT3 biosynthesis of IBMP, is produced in the pulp of the berry complicating the factors that influence IBMP concentration. Our results raise questions that require additional research to clarify this relationship of transcript abundance to IBMP concentration, including determination of the rates of biosynthesis and catabolism, enzyme activities, volatilization of IBMP from the berry, as well as the concentrations of substrates for the enzymes involved. There are a number of other transcriptomic ripening studies in grapes and other fruit species. Many of these have compared broad developmental stages with partial genome microarrays. One study compared transcriptomic responses of the lates stages of ripening of whole berries of Chardonnay. This study used a different microarray platform with only about half of the genome represented on the array. In this study, 12 genes were found to be differentially expressed in each of the 3 different stages investigated. There were approximately another 50 genes that were differentially expressed at one stage versus another. Several genes were proposed as good candidates for markers of ripeness and these were also examined in Cabernet Sauvignon berries using qPCR. Several of these candidate genes are consistent with our results in the present study. They include CCD4a , hydroponic nft channel a late embryogenesis abundant protein , a dirigent-like protein , and an S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase .

Of these, the transcript expression of SAMT was found to be temperature insensitive. Like the previous study, the present study focused on very close stages in the mature berry when fruit flavors are known to develop. In contrast to the previous study on Chardonnay, there were massive changes in the transcript abundance in hundreds of GO categories over this narrow window of ripening. This may in part be due to using six biological replicates rather than the standard three, which probably improved the detection of significantly changing transcripts. In addition, we used a different threshold level for statistical significance and an improved microarray platform, which was able to detect double the number of transcripts. In the present study, many differences were found between the skin and the pulp, °Brix levels and the interaction of tissue and °Brix. Important fruit ripening processes were affected including ethylene signaling, senescence, volatile aroma production, lipid metabolism and cell wall softening. These data indicate that fruit ripening in the late stages of maturity is a very dynamic and active process.Ethylene is involved in climacteric fruit ripening with a CO2 burst preceding the rise in ethylene. In tomato, this occurs at the time the seeds become mature in the mature green fruit stage. At this stage, tomato fruits become sensitive to ethyene and can continue through the ripening stage. Prior to the mature breaker stage, ethylene cannot promote tomato ripening to full ripeness. In non-climacteric fruit, there is no respiratory burst of CO2 and the ripening of most non-climacteric fruits was thought not to respond significantly to an extra application of ethylene. However, recently some non-climacteric fruit such as strawberry, bell pepper and grape have been found to produce a small amount of ethylene and appear to have responses to ethylene at certain stages.

In the study of grapes, this peak was observed just before the start of veraison, followed by decreases in ethylene concentrations for several weeks afterwards; the late mature stages of ripening were not examined. Ethylene action is dependent upon ethylene concentration and ethylene sensitivity or signaling. In this study, there were clear and significant changes in transcript abundance of genes involved in ethylene signaling and biosynthesis in the late stages of berry ripening. Seeds become fully mature at this time . Perhaps there is a signal from the seeds when they become mature that allows the fruit to ripen and senesce? Perhaps small amounts of ethylene are produced or there is a change in sensitivity to ethylene? Seymour et al. suggested the response of EIN3 might be a common signaling mechanism for both climacteric and non-climacteric fruit. The responses of VviEIN3 in this study and in a pepper fruit ripening study are consistent with this hypothesis. In addition, the transcript abundance of VviEIN3 in grape is very responsive to ethylene and the ethylene inhibitor, MCP. There are many other factors other than fruit development that can influence ethylene signaling. Could chilling of the fruit or other aspects of the processing of the grapes influence these responses? Could there be some influence of other abiotic or biotic stresses? These are questions that can only be addressed in future studies with additional experiments that are designed to answer these questions.Hydroxycinnamic acid amides are a group of plant secondary metabolites found in a wide range of plant species. Many studies have identified critical roles that HCAAs play in plant growth and developmental processes, including cell division, cytomorphogenesis, flowering, cell wall cross-linking, tuberization, and stress responses.

These compounds are antioxidants and effective free radical scavengers with anticarcinogenic, antihypertensive, antimicrobial, and other potentially therapeutic activity of significant benefit to human and animal health. Due to the diversity of carbon skeletons, HCAAs can be divided into many categories such as the polyamine conjugates hydroxycinnamoylspermidine , −spermine , and -putrescine . HCSpd and HCPut are predominant in the plant kingdom, while only a few plants are rich in HCSpm compounds . HCSpm, rare in nature, exhibit unique health benefits. For instance, N1 , N14-bis spermine is a major compound that confers the hypotensive and antiparasitic activities in fruit of the Chinese medicinal species Lycium chinense. Trypanothione reductase, an essential enzyme for survival of pathogenic protozoa such as Leishmania and other trypanosomes, is inhibited by afourfold lower concentration of Kukoamine A compared with its spermidine counterpart [N1 , N10-bis -spermidine]. Kukoamine A also shows anticancer activity and attenuates insulin resistance and fatty liver disease. In addition, N1 -coumaroylspermine, but not N1 -coumaroylspermidine, is found to efficiently inhibit mammalian and crayfish neuroreceptors in vitro, an ability of great interest for pest management as well as pain management.Like most solanaceous plants that have been phytochemically analyzed, Solanum fruit crops such as eggplant are rich in HCAAs with spermidine or putrescine, but not spermine, as the polyamine moiety. However, HCSpms possess medicinal properties distinct from those of the other, much more common HCAAs. Although HCSpms have been known for a few decades as rare plant HCAAs of potential therapeutic value, the committed enzyme catalyzing the condensation of hydroxycinnamoyl-CoA with spermine has not been identified so far. In this study, we identified and characterized SpmHT, a spermine exclusive HT from S. richardii, a wild eggplant relative found in Africa.Structurally SpmHT shares linear similarities to other polyamine hydroxycinnamoyl transferase family enzymes, such as spermidine HT and putrescine HT . However, SpmHT has two unique features compared with other HTs. First, SpmHT has the highest activity among all known polyamine HTs, nft growing system as its Vmax is more than 5, 7, 14, 21, and 84 times that of SbHCT, SrSHT, AtSDT, AtSCT, and CcHCT, respectively. Second, SpmHT only utilizes spermine as acyl acceptor. Several catalytic-activityrelated residues have been identified in HTs by mutagenesis, such as Thr-36 and Ser-38 in SbHCT. These two residues are specifically involved in hydroxycinnamoyl moiety binding. The positions corresponding to Thr-36 and Ser-38 are replaced by Leu in SpmHTs, and by Val and Thr in SHTs . Whether these two substitutions in SpmHT have an impact on the catalytic activity is worth further study. As far as the polyamine moiety binding, molecular docking indicates that SpmHT prefers spermine to spermidine based on FullFitness and cluster formation. Three residues involved in the formation of hydrogen bonds are highly conserved in all putative SpmHTs, whereas the corresponding sites in SrSHT are substituted by distinct residues in SrSHT. Further structural study is needed to address how SpmHT specifically selects the acceptor substrate and why it has high enzyme activity.

Our previous analyses show that HCSpms in S. richardii fruit are mainly di- or tri- hydrocaffeoyl acylated spermine . However in vitro studies exhibited a specific monohydrocaffeoyl acylation of spermine by SpmHT. It is not clear whether SpmHT can use di- or tri- hydrocaffeoyl CoA as the substrate in planta. Another possibility is that monosubstituted spermine conjugate may be an acyl acceptor for a second hydroxycinnamoyl transfer by another acyltransferase. In the case of two native tobacco HTs , NaDH29 mediates the initial acylation step specifically on Spd and is not able to perform the second acylation. Further acylation is committed by NaCV86 to act on monoacylated spermidines. Further elucidation requires structural or mutagenesis studies and in vivo functional analysis. SpmHT is highly expressed in fruit of S. richardii but barely in any tissue of S. melongena or other relatives we profiled. Therefore, SpmHT has been selectively silenced in eggplant and many other plants, suggesting that the reduced expression of SpmHT in eggplant fruit would represent an interesting model to investigate molecular mechanism of eggplant phytochemical evolution during the domestication process. High expression of SpmS ensures the sufficiency of Spm for HCSpm synthesis in S. richardii fruit. In contrast, there is almost no expression of SpmS in S. melongena fruits. Hence HCAA composition in S. richardii might be achieved through coordinated expression of HCAA structural genes SpmHT and SpmS. Their coordinated expression may be regulated by some master regulators. MYBs have been shown in other species to regulate HCT expression. For example, in Nicotiana attenuata, MYB8 controls phenolamide levels by directly activating the transcription of three polyamine hydroxycinnamic acid transferases. Since then, they have been similarly implicated in both monocots and eudicots such as strawberry. A recent study showed that a positive regulator ORA59 could bind to the promoter of an Arabidopsis agmatine coumaroyl transferase and enabled its expression and HCAAs biosynthesis to respond to simultaneous activation of the JA/ET signaling pathways. It would be interesting to identify the master regulators involved in SpmHT biosynthesis in order to activate the pathway and study the function of SpmHT. High levels of SpmS expression suggest that abundant Spm is synthesized in S. richardii fruit. This is unusual because Spm is synthesized at lower levels than Spd in most plants. Generally speaking, Spd is thought to contribute to higher vegetative growth, less shriveling , and longer life span in transgenic plants over expressing SpdS, while elevated Spm promotes abiotic and biotic stress tolerance by inducing the expression of defense genes in plants . Hence, it appears that Spd is largely implicated in developmental processes, whereas Spm is more likely involved in stress response. However, excessive Spm maycause abnormal development and consequently there must be tight regulation of polyamine homeostasis for normal growth of plants.The pineapple Merr is the 3rd most important fruit traded globally, but it is susceptible to chilling injury . Chilling injury is a complex physiological disorder that occurs in tropical and sub-tropical fruit, including pineapple, after exposure to low temperatures . Chilling may occur in the field in winter grown fruit causing pre-harvest chilling injury, or after harvest when fruit are stored at low temperatures, i.e., post harvest chilling injury; PCI . Losses of up to 80% have been attributed to PCI of pineapples after storage at 10 ◦C , which reduces wider market ability of the fruit. Because of the magnitude of the problem presented by PCI, the focus of this study was limited to this phenomenon and did not include pre-harvest chilling injury.The severity of PCI depends on the temperature to which fruit is exposed and the duration of exposure. PCI symptoms in pineapple are initiated after 1–3 weeks storage at temperatures below 12 ◦C followed by a transfer to room temperature . Symptoms mainly occur in the fleshy region adjacent to the core  and the earliest manifestation is tissue translucency symptoms . As the symptoms intensify, browning or blackening of the tissue becomes noticeable. The resulting fruit damage, also called “internal browning” , “black heart,” or “endogenous brown spot,” occurs internally with no external signs of injury . IB-susceptible pineapple cultivars belonging to the ‘Queen’ group show symptoms within 1–2 weeks after storage at temperatures below 10–12 ◦C . The tissue first turns translucent, then brown, in both the core and the fleshy tissue of the fruit .

At a fruit jam manufacturing facility in Manchester, England, cleaning hoses in the fruit room were identified as one of the highest end uses of water in the facility . The new nozzles and training cost only £100 , but resulted in savings of £3,000 to £4,000 per year . The simple payback period for this measure was less than two weeks.Similarly, Harvest FreshCuts was able to reduce the water it uses for cleaning by 10% through the installation of efficient high pressure spray nozzles on hoses, regular hose and nozzle maintenance, and operator training . Clean equipment immediately after use. Waiting too long to clean equipment can allow product residues to become dry and crusty and harder to remove, requiring more water consumption in the cleaning phase. Processing equipment should be immediately cleaned after production has stopped to minimize the water necessary for cleaning . Optimization of clean-in-place performance. Clean-in-place processes should be programmed to use only enough water and detergent to perform the desired cleaning task at a particular piece of equipment. Dry cleaning prior to clean-in-place cycles can further reduce the minimum amount of water and detergent needed . An environmental assessment at Harvest FreshCuts determined that by introducing a cleaning system that ensured accurate water and chemical usage during the cleaning cycle, blueberry grow pot the volume of internally recycled water could be increased by 40% . Pigging. A pig is a solid plug or ball that is pushed through a pipe to remove deposits adhering to pipe walls. Pigging can be performed instead of rinsing pipes with water to reduce water consumption, where the piping system is amenable to pigging .

At Nelsons of Aintree, a jam manufacturer based in the United Kingdom, a pigging system was installed to clean a long pipeline used for transporting jam. Previously, cleaning the pipeline used large volumes of water and flushed most of the jam residues into a drain. In the new system, rubber pigs made from food grade rubber are propelled through the pipe using compressed air and stopped at the other end of the pipe by a bar that stops the pig but allows jam to pass. The pig is returned by switching the direction of the compressed air via a valve. The pigging system saved the company £105,280 per year by reducing water use, effluent discharge volumes, and energy consumption and another £134,780 per year in avoided product losses . Reportedly, the system allowed the company to save 173 tons of jam per year while reducing water use by around 528,000 gallons per year. Low pressure foam cleaning. Traditionally, walls, floors, and equipment are cleaned using brushes, high pressure spray hoses, and detergents. Low pressure foam cleaning methods, in which cleaning foam is sprayed on surfaces and allowed to settle for 10 to 20 minutes before rinsing with low pressure water, can save both water and energy compared to high pressure cleaning methods . However, this method does not provide scouring ability and thus might not be a feasible replacement for all high pressure cleaning applications.Control of volume in clean-in-place processes. The control of water flows for burst rinsing and clean-in-place processes should be based on required water volume, not a pre-determined time, to reduce cleaning water quantities . Pre-soaking of floors and equipment. An effective means of reducing water consumption in cleaning is to pre-soak soiled surfaces on floors and open equipment prior to cleaning. Pre-soaking can be effective at loosening dirt and hardened food residues so that less water is required in the actual cleaning operations .In the conventional method of once-through water use , fresh water is used once for process and cleaning applications and any water not contained in the final product is then discharged into the wastewater stream.

Although once-through water use methods are increasingly less common in modern fruit and vegetable processing facilities, they represent the most inefficient methods of using water and should be avoided wherever possible. Preferably, water should be recovered and reused or recycled within the facility to reduce fresh water purchasing and treatment costs while also reducing the volume and associated costs of wastewater treatment and disposal. According to Raghupathy , at least 50% to 60% of water in typical food processing facilities can be recovered for reuse. However, the extent to which fresh water use can be reduced via water reuse and recycling measures in any fruit and vegetable processing facility will ultimately depend on product hygiene considerations.In general, recycling is feasible and practiced commonly in the U.S. food processing industry in the first two applications but generally not practiced in the third application due to hygienic concerns. Where feasible, the elimination of once-through water use can lead to significant water savings, as illustrated by the following case studies. At the Gangi Brothers Packing Company, a canned tomato product manufacturer in San Jose, California, water is used in fluming tomatoes from trucks, tomato rinsing, vacuum pump seals, boiler makeup, and process cooling. In 1989, the company implemented an aggressive water efficiency program, which included the recycling of flume water and the installation of evaporative cooling towers to recycle cooling water. Reportedly, the company reduced its water consumption by 94 million gallons per year, which led to savings of around $130,000 per year . The reported payback period on the equipment and modifications was less than one year. Stahlbush Island Farms, a grower, canner, and freezer of fruits and vegetables in Corvalis, Oregon, reduced its consumption of water by more than 50% through innovative water recovery and recycling systems. Water is pumped from wells at a temperature of about 55° F, where it is quickly used to cool hot pumpkin puree. Next, the water passes through a second heat exchanger, where it cools oil from the facility’s refrigeration system compressors. When the water leaves the second heat exchanger, it has been heated to around 100° F.

The warm water is then pumped to a surge tank, where it is used in one of four different applications: to wash pumpkins as they enter the processing plant, to clean food processing equipment, for condenser water in the facility’s refrigeration cycle, and for boiler makeup water . As an additional benefit, the recycled water is used to provide warm boiler makeup water and to preheat the washed pumpkins, saving energy. Listed below are some of the most significant opportunities for water recovery, reuse, and recycling applicable to fruit and vegetable processing facilities.Reuse of washing water. In the initial washing of fruits and vegetables, a large volume of water is often necessary and the concentration of dirt in the wastewater exiting the process is typically low. In many instances, a recirculation system can be installed to maintain an acceptable concentration of dirt in the wash water while reducing fresh water inputs. A basic recirculation system consists of a strainer or filter to remove solids and a pump for circulating water back to the washing process . For wash water with high dirt concentrations, a flotation unit or centrifugal separator can be added to help remove solids. Additionally, ultraviolet or ozone treatment modules can be added to reduce bacterial loads, where needed. Cooling towers. Once-through cooling systems can be replaced by cooling towers, which continuously recycle cooling water and lead to significant water savings. The U.S. DOE estimates that to remove the same heat load, once-through cooling systems can use as much as 40 times more water than a cooling tower . In a cooling tower, circulating warm water is put into contact with an air flow, hydroponic bucket which evaporates some of the water. The heat lost by evaporation cools the remaining water, which can then be recirculated as a cooling medium. For example, cooling towers can be used to recirculate water from evaporative can coolers in the canning process, with recycling occurring continuously until the water no longer meets cleanliness standards .Recycling of final rinse water. Final rinsing is done to remove residues of detergents from the equipment after it has been cleaned. The final rinse water, while not suitable for additional final rinsing applications, can be recovered and used for initial rinsing or intermediate rinsing purposes rather than being discharged to the wastewater stream . Recycling of evaporator condensate. Depending on the quality of condensate reclaimed from products in evaporation processes, condensate water can be reused for other low-grade facility applications such as equipment pre-rinsing and surface pre-soaking. Additionally, condensate recovery systems can be fitted with heat exchangers such that hot condensate can be used for pre-heating the evaporation process input streams, which saves energy . Segregation of wastewater systems. When all facility wastewater streams are combined into a common wastewater flow, opportunities for recovering and recycling the wastewater streams with reclaimable water are lost. Where feasible, the use of separate process wastewater systems should be considered to maximize opportunities for water recovery and recycling. For example, in 1993 a UK based snack food company performed a facility audit to determine if water savings could be realized if process wastewater streams were segregated prior to on-site treatment. The company found that by segregating its potato wash water, hot starch water, and cold starch water streams for separate recovery and treatment, its water consumption could be reduced by 19% .

The potato wash water was reused after grit removal and the cold starch water was recycled after good quality starch was recovered. The annual savings in water supply costs were estimated at £90,000 .Membrane filtration. Membrane filtration technologies have been applied in many industries to clean wastewater prior to disposal and to recover water for recycling in various facility and process applications. Membrane systems used in wastewater treatment at fruit and vegetable processing facilities have been documented to reduce freshwater intake and effluent by as much as 85% . The potential barriers to implementation include relatively high capital costs, as well as the need for specific membranes for specific applications . At the Tri Valley Growers’ Oberti Olive facility in Madera, California, reverse osmosis and ultra-filtration membrane systems were installed to treat the facility’s well water, flotation brine, oil mill slurry, yeast broth, and biotower water. The membrane systems reportedly reduced the company’s freshwater intake and effluent discharge by 80% to 85%, allowed for the recovery of salt from brine water, allowed for the recovery of solids for sale as animal feed, and reduced land use by evaporation ponds by 85% . At the Michigan Milk Producers Association facility in Ovid, Michigan, a reverse osmosis membrane filtration system was installed to concentrate organic impurities in evaporator condensate. The filtered hot condensate water is reused for clean-in-place water, tank wash down water, and boiler makeup water. The reported benefits include a reduction in well water consumption and wastewater discharges of 100,000 to 150,000 gallons per day, a reduction in boiler and wash water treatment costs of $6,000 to $8,000 per month, and a reduction in scale buildup on pipes . Hydrocyclones. For wastewater streams with significant solids content, such as heavily soiled wash water, hydrocyclones can be used to separate out solids and reclaim water for use in other facility applications. Such systems can often have three major benefits. First, a significant amount of water can be recovered and recycled within a facility, reducing the necessary purchases of fresh water. Second, because wastewater ultimately has less solids content, wastewater disposal costs are often reduced. Third, recovered solids can often be recycled as animal feed, mulch, or agricultural additives. At the Smith Snack Food Company, the largest manufacturer of potato- and corn-based snack foods in Australia, a hydrocyclone system was installed in 1997 to reduce the solids content in wastewater streams at the company’s Adelaide facility. Hydrocyclones were installed on the facility’s corn and potato washing lines, with solids being collected in a sludge tank and reclaimed water being recycled back into the initial washing processes for potatoes and corn. The system reportedly reduced water consumption in the washing processes by more than 80% while also saving the company around $130,000 per year in reduced wastewater disposal costs . The simple payback period was estimated at just five weeks. Recycling of can cooling water. When can cooling water is not recirculated, it can be recovered and used for the initial washing of incoming products, as a rinse for caustic peeling processes, in canning belt lubrication, and in miscellaneous facility cleanup operations .Recycling of blanching and cooking water.

Common energy efficiency measures for industrial compressed air systems are discussed below. Additionally, a number of measures that are applicable to refrigeration system compressors and motors are also applicable to compressed air systems.The most common areas for leaks are couplings, hoses, tubes, fittings, pressure regulators, open condensate traps and shut-off valves, pipe joints, disconnects, and thread sealants. The best way to detect leaks is to use an ultrasonic acoustic detector, which can recognize the high frequency hissing sounds associated with air leaks. Leak detection and repair programs should be ongoing efforts. In 1994, Mead-Johnson Nutritionals, a manufacturer of infant formula and adult nutritional supplements, implemented a compressed air system improvement project at its plant in Evansville, Indiana. Energy efficiency measures included the introduction of a monitoring system, the installation of new compressors, and the repair of leaks. The improved compressed air system of this plant functioned so efficiently that only two-thirds of the compressed air capacity had to be kept online. The company saved $102,000 per year in compressed air system energy costs with a payback period of just over 2.5 years. Additionally, the project helped the plant avoid the purchase of a new compressor . Turning off unnecessary compressed air. Equipment that is no longer using compressed air should have the air turned off completely. This can be done using a simple solenoid valve. Compressed air distribution systems should be checked when equipment has been reconfigured to ensure that no air is flowing to unused equipment or to obsolete parts of the compressed air distribution system. Modification of system in lieu of increased pressure. 

For individual applications that require a higher pressure, instead of raising the operating pressure of the whole system, nft hydroponic system special equipment modifications should be considered, such as employing a booster, increasing a cylinder bore, changing gear ratios, or changing operation to off peak hours.Based on numerous industrial case studies, the average payback period for replacing compressed air with other applications is estimated at 11 months . Improved load management. Because of the large amount of energy consumed by compressors, whether in full operation or not, partial load operation should be avoided. For example, unloaded rotary screw compressors still consume 15% to 35% of full-load power while delivering no useful work . Air receivers can be employed near high demand areas to provide a supply buffer to meet short-term demand spikes that can exceed normal compressor capacity. In this way, the number of required online compressors may be reduced. Multi-stage compressors theoretically operate more efficiently than single-stage compressors. Multi-stage compressors save energy by cooling the air between stages, reducing the volume and work required to compress the air. Replacing single-stage compressors with two-stage compressors typically provides a payback period of two years or less . Using multiple smaller compressors instead of one large compressor can save energy as well. Large compressors consume more electricity when they are unloaded than do multiple smaller compressors with similar overall capacity. An analysis of U.S. case studies shows an average payback period for optimally sizing compressors of about 1.2 years . In June 2004, the Canandaigua Wine Company upgraded the compressed air system at its winery in Lodi, California. Before the project began, the winery was served by two 125 hp rotary screw compressors that operated at full load only during the 3-month fall grape crushing season.

During the rest of the year, however, the compressors were operated at part-load, which wasted energy. The company opted to install a 75 hp variable-speed compressor, which could be used to satisfy facility demand during the off-season while also providing supplemental power to the two 125 hp units during the fall crush season. Additionally, the company installed a new compressor control system, additional storage, and started a leak reduction campaign. The total energy savings attributable to the upgrade were estimated at 218,000 kWh per year, saving the company $27,000 annually . The simple payback period was estimated at 1.2 years. Similarly impressive savings were realized with a compressor upgrade at a Sara Lee bakery in Sacramento, California, in 2004. Prior to the upgrade, the company used one 100 hp and two 150 hp rotary screw compressors in its compressed air system. After the upgrade, the company used the 100 hp fixed-speed unit as its base compressor and a new 100 hp ASD compressor for variable loads. The project reduced annual facility energy consumption by 471,000 kWh and annual energy costs by around $40,000, while also saving the company$10,000 per year in avoided maintenance costs . The reported payback period was just 6.5 months. Pressure drop minimization. An excessive pressure drop will result in poor system performance and excessive energy consumption. Flow restrictions of any type in a system, such as an obstruction or roughness, results in higher operating pressures than is truly needed. Resistance to flow increases the drive energy on positive displacement compressors by 1% of connected power for each 2 psi of differential . The highest pressure drops are usually found at the points of use, including undersized or leaking hoses, tubes, disconnects, filters, regulators, valves, nozzles, and lubricators , as well as air/lubricant separators on lubricated rotary compressors and after-coolers, moisture separators, dryers, and filters .

Minimizing pressure drop requires a systems approach in design and maintenance. Air treatment components should be selected with the lowest possible pressure drop at specified maximum operating conditions and best performance. Manufacturers’ recommendations for maintenance should be followed, particularly in air filtering and drying equipment, which can have damaging moisture effects like pipe corrosion. Finally, the distance the air travels through the distribution system should be minimized. Audits of industrial facilities found that the payback period is typically shorter than 3 months for this measure . Inlet air temperature reduction. If airflow is kept constant, reducing the inlet air temperature reduces the energy used by the compressor. In many plants, it is possible to reduce the inlet air temperature to the compressor by taking suction from outside the building. As a rule of thumb, each temperature reduction of 5°F will save 1% compressor energy . A payback period of two to five years has been reported for importing fresh air . In addition to energy savings, compressor capacity is increased when cold air from outside is used. Industrial case studies have found an average payback period for importing outside air of less than 1.7 years , but costs can vary significantly depending on facility layout.Yasama Corporation U.S.A., a manufacturer of soy sauce, installed new compressor system controls at its Salem, Oregon, facility in 2004. Previously, the company ran its three compressors using inefficient individual load/unload controls. Additionally, the company added two 2,200 gallon air storage receivers to help handle the facility’s short-term peak loads. Under the new control strategy, the three compressors were sequenced to run most efficiently, leading to annual energy savings of 100,000 kWh and annual electricity savings of $5,100 . Additionally, the new control system allowed the company to better manage the total operating hours of each compressor as well as the number of starts per unit per hour, helping to reduce compressor wear and tear. In addition to energy savings, the application of controls can sometimes eliminate the need for some existing compressors, allowing extra compressors to be sold or kept for backup. Alternatively, hydroponic nft system capacity can be expanded without the purchase of additional compressors.Properly sized pipe diameters. Increasing pipe diameters to the greatest size that is feasible and economical for a compressed air system can help to minimize pressure losses and leaks, which reduces system operating pressures and leads to energy savings. Increasing pipe diameters typically reduces compressed air system energy consumption by 3% . Further savings can be realized by ensuring other system components are properly sized. H.B. Reese, a subsidiary of the Hershey Foods Company, overhauled the compressed air system piping network at its Hershey, Pennsylvania, facility in 1996. The plant modified and replaced undersized components such as filters, lubricators, fittings, and hoses, which lowered the minimum system operating pressure from 85 psi to 75 psi . Heat recovery. As much as 90% of the electrical energy used by an industrial air compressor is converted into heat. In many cases, a heat recovery unit can recover 50% to 90% of this available thermal energy and apply it to space heating, process heating, water heating, makeup air heating, boiler make-up water preheating, and heat pump applications .

It has been estimated that approximately 50,000 Btu/hour of recoverable heat is available for each 100 cfm of compressor capacity . Payback periods are typically less than one year . Heat recovery for space heating is not as common with water-cooled compressors because an extra stage of heat exchange is required and the temperature of the available heat is somewhat low. However, with large water-cooled compressors, recovery efficiencies of 50% to 60% are typical . Natural gas engine-driven air compressors. Gas engine-driven air compressors can replace electric compressors with some advantages and disadvantages. Gas engine-driven compressors are more expensive and can have higher maintenance costs, but may have lower overall operating costs depending on the relative costs of electricity and gas. Variable-speed capability is standard for gas-fired compressors, offering a high efficiency over a wide range of loads. Heat can be recovered from the engine jacket and exhaust system. However, gas engine-driven compressors have some drawbacks: they need more maintenance, have a shorter useful life, and sustain a greater likelihood of downtime. According to Galitsky et al. , gas engine-driven compressors currently account for less than 1% of the total air compressor market. Ultra Creative Corporation, a U.S. manufacturer of specialty plastic bags, installed gas engine-driven compressors in its plant in Brooklyn, New York. The initial costs were $85,000 each for two 220 hp units and $65,000 for one 95 hp unit. The company reported savings of $9,000 in monthly utilities .Energy-efficient system design. By sizing equipment properly and designing energy efficiency into a new facility, fruit and vegetable processors can minimize the energy consumption and operational costs of HVAC systems from the outset. This practice often saves money in the long run, as it is generally cheaper to install energy efficient HVAC equipment at building construction than it is to upgrade an existing building with an energy-efficient HVAC system later on, especially if those upgrades lead to production downtime. Recently, Mission Foods, a California manufacturer of specialty Mexican foods, worked with Southern California Edison to design its new production facility in Rancho Cucamonga to be as energy efficient as possible. The new facility had 50,000 square feet of office space, 125,000 square feet of manufacturing space, and 134,000 square feet of warehouse space. Mission Foods chose to install energy-efficient technologies for its HVAC systems and lighting systems, room occupancy sensors that turned off lights automatically, low-emissivity windows that reduced building heat gain, and skylights that provided natural lighting. The total project allowed the company to reduce the electricity consumption of its new facility by roughly 18% compared to its existing facilities, leading to annual energy savings of over $300,000 per year . Recommissioning. Before replacing HVAC system components to improve energy efficiency, the possibility of HVAC system recommissioning should be explored. Recommissioning is essentially the same process as commissioning, but applied to a building’s existing HVAC, controls, and electrical systems . Commissioning is the process of verifying that a new building functions as intended and communicating the intended performance to the building management team. This usually occurs when a new building is turned over for occupancy. In practice, commissioning costs are not included in design fees and often compete with other activities. As a result, commissioning is seldom pursued properly. It is critical that the building is commissioned to ensure that energy performance and operational goals are met.Recommissioning involves a detailed assessment of existing equipment performance and maintenance procedures for comparison to intended or design performance and maintenance procedures to identify and fix problem areas that might be hampering building energy efficiency. Recommissioning can be a cost-effective retrofit in itself, sometimes generating more savings than the cost of the retrofit measure. For example, recommissioning may help avoid the need to install new or additional equipment, leading to savings in capital investments.

Further research on the economics of all measures—as well on as their applicability to different production practices—is needed to assess their cost effectiveness at individual plants. This Energy Guide also presents a brief overview of selected emerging energy-efficient technologies, which have recently been developed or commercialized and hold promise for reducing energy use in the U.S. fruit and vegetable processing industry in the near future. While the focus of this Energy Guide is on energy efficiency improvement measures, a chapter on basic, proven measures for plant-level water efficiency is also provided in recognition of the importance and rising costs of water as a resource in the U.S. fruit and vegetable processing industry. Water savings can also lead to energy savings through reduced demand for water heating, treatment, and pumping.To enable easy access to information, this Energy Guide is organized into chapters that focus on specific areas of opportunity for energy and water efficiency. Chapters 6 through 12 are focused on cross-cutting energy efficiency measures, which are defined as energy efficiency measures that are applicable across all manufacturing industries. Table 5.1 summarizes the cross-cutting energy efficiency measures presented in this Energy Guide and the respective chapters in which the measure descriptions appear. After a brief overview of corporate energy management programs in Chapter 6, this Energy Guide focuses on the following cross-cutting industrial systems, which are of particular importance to the U.S. fruit and vegetable processing industry: steam systems, motors and pumps, compressed air systems, large round pot refrigeration systems, building systems and self generation.Changing how energy is managed by implementing an organization-wide energy management program is one of the most successful and cost-effective ways to bring about energy efficiency improvements.

Continuous improvements to energy efficiency typically only occur when a strong organizational commitment exits. A sound energy management program is required to create a foundation for positive change and to provide guidance for managing energy throughout an organization. Energy management programs help to ensure that energy efficiency improvements do not just happen on a one-time basis, but rather are continuously identified and implemented in an ongoing process of continuous improvement. Without the backing of a sound energy management program, energy efficiency improvements might not reach their full potential due to lack of a systems perspective and/or proper maintenance and follow-up. In companies without a clear program in place, opportunities for improvement may be known but may not be promoted or implemented because of organizational barriers. These barriers may include a lack of communication among plants, a poor understanding of how to create support for an energy efficiency project, limited finances, poor accountability for measures, or organizational inertia to changes from the status quo. Even when energy is a significant cost, many companies still lack a strong commitment to improve energy management. The U.S. EPA, through the ENERGY STAR program, works with leading industrial manufacturers to identify the basic aspects of effective energy management programs.17 The major elements in a strategic energy management program are depicted in Figure 6.1. Other environmental management frameworks, such as ISO 14001, can be used to complement energy management programs to ensure optimal organizational management of energy.

One ENERGY STAR partner noted that using energy management programs in combination with the ISO 14001 program has had a greater impact on conserving energy at its plants than any other strategy. A successful program in energy management begins with a strong organizational commitment to continuous improvement of energy efficiency. This involves assigning oversight and management duties to an energy director, establishing an energy policy, and creating a cross-functional energy team . Steps and procedures are then put in place to assess performance through regular reviews of energy data, technical assessments, and bench marking. From this assessment, an organization is able to develop a baseline of energy use and set goals for improvement. Performance goals help to shape the development and implementation of an action plan.An important aspect for ensuring the success of the action plan is involving personnel throughout the organization. Personnel at all levels should be aware of energy use and goals for efficiency. Staff should be trained in both skills and general approaches to energy efficiency in day-to-day practices. Some examples of simple tasks employees can do are outlined in Appendix B. In addition, performance results should be regularly evaluated and communicated to all personnel and high achievement should be rewarded and recognized.For example, ConAgra Foods has recognized outstanding employee contributions to energy efficiency as part of its corporate Sustainable Development program since 1993. Each year, several ConAgra production facilities are given a monetary award for outstanding plant initiated projects that led to energy savings and other environmental improvements.

The monetary awards are used by the production facilities as charitable donations to their communities for local sustainability projects. In addition to providing its employees with recognition and incentives for continuous improvement, ConAgra’s Sustainable Development program has also reduced facility operating expenses by over $60 million since 2000 . Evaluating progress on the action plan involves a regular review of both energy use data and the activities carried out as part of the action plan. Information gathered during the formal review process helps in setting new performance goals and action plans, and in revealing best practices. Once best practices are established, the goal of the cross-functional energy team should be to replicate these practices throughout the organization. Establishing a strong communication program and seeking recognition for accomplishments are also critical steps, as both areas help to build support and momentum for future activities. A quick assessment of an organization’s efforts to manage energy can be made by comparing its current energy management program against the ENERGY STAR Energy Program Assessment Matrix provided in Appendix C. Frito-Lay, a manufacturer of snack foods headquartered in Plano, Texas, implemented a comprehensive corporate energy management program in 1999 that has led to energy savings of 21% across its 34 U.S. facilities and saved the company more than $40 million in energy costs to date . Key components of this plan include: the designation of three tiers of energy management personnel , capital budgets that are designated exclusively for energy efficiency improvements, annual energy budget target setting for each site with weekly performance tracking, and an annual energy summit for continuing education, sharing of success stories between facilities, and awards for top performers . Internal support for a business energy management program is crucial; however, support for business energy management programs can come from outside sources as well. Some utility companies work together with industrial clients to achieve energy savings in both existing facilities and in the design of new facilities. Recently, Mission Foods, a California manufacturer of specialty Mexican foods, worked with Southern California Edison to design its new production facility in Rancho Cucamonga to be as energy efficient as possible. By employing energy-efficient technologies for motors, HVAC systems, compressors, and lighting throughout the facility, Mission Foods was able to reduce the electricity consumption of its new facility by roughly 18% compared to its existing facilities. Annual energy savings of over $300,000 per year were achieved . Facility audits can be another particularly effective form of outside support. In a recent audit carried out by U.S. DOE Industrial Assessment Center staff at an Odwalla Juice Company facility in Dinuva, California, big round plant pot energy efficiency opportunities were identified that would reduce annual energy costs by $268,000 and annual energy usage by 15% with an average payback period of just 20 months .In forming an energy team, it is necessary to establish the organizational structure, designate team members, and specify roles and responsibilities. Senior management needs to perceive energy management as part of the organization’s core business activities, so ideally the energy team leader will be someone at the corporate level who is empowered by support from senior-level management. The energy team should also include members from each key operational area within an organization and be as multi-disciplinary as possible to ensure a diversity of perspectives. It is crucial to ensure adequate organizational funding for the energy team’s activities, preferably as a line item in the normal budget cycle as opposed to a special project. Prior to the launch of an energy team, a series of team strategy meetings should be held to consider the key initiatives to pursue as well as potential pilot projects that could be showcased at the program’s kickoff. The energy team should then perform facility audits with key plant personnel at each facility to identify opportunities for energy efficiency improvements. As part of the facility audits, the energy team should also look for best practices in action to help highlight success stories and identify areas for inter-plant knowledge transfer.

A key function of the energy team is to develop mechanisms and tools for tracking and communicating progress and for transferring the knowledge gained through facility audits across an organization. Examples of such mechanisms and data tools include best practice databases, facility bench marking tools, intranet sites, performance tracking scorecards, and case studies of successful projects. Corporate energy summits and employee energy fairs are also effective means of information exchange and technology transfer. To sustain the energy team and build momentum for continuous improvement, it is important that progress results and lessons learned are communicated regularly to managers and employees and that a recognition and rewards program is put in place. A checklist of key steps for forming, operating, and sustaining an effective energy management team is offered in Appendix D. Energy monitoring systems may include energy sub-metering at the component, equipment, or process level and can be used to track various end uses of energy over time for energy efficiency improvement analysis. These systems can play a key role in alerting energy teams to problem areas and in assigning accountability for energy use within a facility. Furthermore, energy monitoring systems can provide useful data for corporate greenhouse gas accounting initiatives. Energy monitoring and metering systems can also help companies participate in emergency demand response programs, in which utility companies provide financial incentives to customers who reduce their energy loads during peak demand times. S. Martinelli and Company, an apple juice manufacturer based in Watsonville, California, installed an energy monitoring system that provided it with real-time data on peak demand and energy consumption. This system allowed them to participate in a demand response program of their local utility. S. Martinelli also uses their system to verify electric and natural gas bills against their actual measured use as a cost control measure, as well as to track facility performance in system optimization efforts .Boiler process control. Flue gas monitors maintain optimum flame temperature and monitor carbon monoxide , oxygen, and smoke. The oxygen content of the exhaust gas is a combination of excess air and air infiltration. By combining an oxygen monitor with an intake airflow monitor, it is possible to detect even small leaks. A small 1% air infiltration will result in 20% higher oxygen readings. A higher CO or smoke content in the exhaust gas is a sign that there is insufficient air to complete fuel burning. Using a combination of CO and oxygen readings, it is possible to optimize the fuel/air mixture for high flame temperature and lower air pollutant emissions. Typically, this measure is financially attractive only for large boilers, because smaller boilers often will not make up the initial capital cost as easily. Several case studies indicate that the average payback period for this measure is around 1.7 years . At Glanbia Foods, a dairy product manufacturer in Lockerbie, Scotland, the installation of a boiler control system reduced annual boiler fuel consumption by 5% . At the J.R. Simplot Company potato processing facility in Caldwell, Idaho, the installation of new burners equipped with process controls and a flue gas trim system led to significant annual savings in natural gas consumption. The Caldwell facility produces approximately 270 million pounds of frozen French fries each year and uses steam in its potato peeling, blanching, and frying operations. In 2003, new burners, flue gas oxygen analyzers, flue gas recirculation ducts, and boiler controls were installed on two boilers during plant outages. Natural gas consumption was reduced by 7.5%, resulting in cost savings of $279,000 per year and a payback period of around 14 months . Reduction of flue gas quantities. 

In addition to running the PLSPM, 1000 bootstraps were performed to obtain statistical significance and confidence intervals of the path coefficients and the R2 values of each latent variable. The path coefficients are the standardized partial regression coefficients , and represent the direction and strength of causal relationships of direct effects. Indirect effects are the multiplied coefficients between the predictor variable and the response variable of all possible paths other than the direct effect . To determine the best path model, the latent variables were combined using our best understanding of biological relationships, and a general model using all data was generated. The paths between LVs were altered until a best-fit model was found. PLS-Predict was then used on the dataset to ensure that the model did not over or under fit the data, and for predictive performance of each manifest variable . This structural model, and not the fit values, was retained for use in predictive modeling of a separate dataset. PLSPREDICT, with the structural model developed as described earlier, was used on a separate dataset to determine the efficacy of the model. Two commercial cultivars, M82 and Lukullus, were used and only the leaf shape values were entered as exogenous variables. The predicted values for each output variable were compared with the actual measured values to determine how well the model predicted these variables.Yield, and fruit BRIX were measured over 14 wk of the growing season . For most cultivars the yield remained at or below 5 kg of fruit per plant at the time of harvest . The exceptions to this were Bloody Butcher, Glacier, Brandywine, Prudens Purple, and Stupice. All had a yield higher than 5 kg per plant, large round pot with Stupice having the highest yield, at c. 20 kg per plant by final harvest . Fruit BRIX remained nearly constant for all cultivars across the growing season , with the exception of ABC Potato Leaf, which had a large increase at time of harvest.

To better quantify fruit quality, BRIX and yield were multiplied to obtain a BY value for each measurement . Bloody Butcher, Glacier, and Stupice all had a BY value > 60, with Stupice reaching near 100 at terminal harvest . The average BY value for harvest weeks was 16.39, while Bloody Butcher, Brandywine, Glacier, and Prudens Purple had an average of c. 23 . Stupice showed the highest deviation with a mean BY value of 37.86 , setting it apart as the highest fruit quality cultivar tested in this study. Stupice maintained a stable BRIX content in its fruit despite the large increase in yield , which resulted in the large increase in overall fruit quality compared with other cultivars. Vegetative traits such as total biomass and leaf area were measured for the growing season as well . Fig. 2 shows the vegetative biomass and leaf area over the course of the growing season, which remain stably linked, indicating that overall leaf area increase contributed to increased biomass of the plant. This trend appears common in heirloom tomatoes but is different in commercial tomatoes, which have determinate growth .Leaf shape was shown to be strongly correlated with fruit BRIX and sugar accumulation in a meta-analysis of an introgression population . How leaf shape contributed to fruit BRIX was unclear, as shape and size of leaves do impact photosynthesis directly , but direct links between leaf shape and fruit quality appear lacking. Here, the heirloom population used displayed a wide array of leaf shapes, from very large and narrow to small and round. To understand if this range of leaf shapes had any impact on the overall fruit quality we measured leaflet shape and size for c. 3733 leaflets. Fig. 4 shows the resultant PCs of all primary leaflets measured and their relationship to traditional shape measures. PC1 contributes 78% of all variation found in the population and is tightly correlated with leaflet size , indicating that size was the largest source of variation among the heirloom leaflets . PC1 was also correlated with solidity , contributing to the slight shape changes seen in this PC . PC2 and PC4, while having no traditional shape measure correlation, indicate the left- and right handedness of the lateral primary leaflets, as these leaflets are mirror images of each other and therefore this measure describes the overall variation in leaf symmetry .

PC3 accounts for 3.8% of all variation, but has a strong correlation with aspect ratio, or the width divided by the length of the leaflet, with an R2 of 0.8 . PC3 therefore represents the roundness or narrowness of the leaflets, one aspect previously shown to be linked to fruit quality . The heirloom cultivars analyzed here were described as ‘potato leaf’, having broader, smoother leaves and typically lack the serration and lobes seen in other tomato varieties . However, despite this they had a wide range of leaf shape and size as illustrated in the leaf shape analysis . The classical potato leaf mutation is caused by a 5 kb transposable element  inserted into the third exon of the C locus . To determine if this locus harbored mutations in the selected lines, a subset of the higher performing cultivars were selected for WGS analysis. Other mutations at the C locus have been described, and cause varied leaf shape . Fig. 5 shows the location of the mutations found in the C locus in these select lines. While the full Rider insertion could not be directly determined as the reference genome lacks this insertion, overhangs on reads in the third exon matched the Rider TE sequence . It is possible that different sizes and fragments of Rider are present in different cultivars, as the length and sequence of the overhangs varied . The identified Rider sequences were present in all but two of the sequenced lines, Prudens Purple and Glacier. No mutations were found in Glacier despite it having a rounder leaflets, although these were smaller in size with higher overall leaf complexity . Prudens Purple had a novel single base-pair substitution in the first exon outside the MYB/SANT conserved domain which results in the amino acid change P42R . We analyzed this mutation using the PROTEIN VARIANCEEFFECT ANALYZER , and found that it is predicted to be deleterious to the protein with a value of 8.454 , predicted to result in either a nonfunctional or partially functional protein . Based on leaf shape analysis, Prudens Purple shows a Potato Leaf like phenotype , although it differs slightly from the classical Potato Leaf shape seen in the reference allele and is reminiscent of the other mutations in C that have varying leaf shapes . These data demonstrate that different mutations in C, coupled with genetic background differences, may give rise to a range of leaf shapes seen among some of these cultivars.Pedigrees would probably inform the overall leaf shape in addition to the source of the C-locus mutations, but were not readily available. To elucidate relationships among these cultivars we used the WGS data from the select cultivars as well as WGS data obtained from the 150 Genomes Project to assemble a phylogeny and perform phylogenetic network analysis . The phylogeny includes several commercial cultivars, commercial heirloom cultivars, Solanum pimpinellifolium, and Solanum lycopersicum var. cerasiforme. ABC Potato Leaf does not appear to cluster with other Potato Leaf heirloom cultivars analyzed here . Stupice, Glacier, and Bloody Butcher are closely related in this phylogeny, corresponding to their often being listed as closely related in popular literature , and congruent with phenotypic similarities they exhibit in fruit size and leaf shape.

Bloody Butcher and Stupice both have the Rider insertion in the third exon at the C locus, while Glacier does not , suggesting the presence of other modifiers to leaf shape, which may have been selected for during the breeding of Glacier. A similar situation is seen in Prudens Purple , which is closely related to Jerry’s German Pink and Green Pearl. While Jerry’s German Pink and Green Pearl carry the Rider insertion at C, a novel single-base-pair substitution in the first exon leading to a deleterious effect on protein function is seen in Pruden’s Purple. Included in the clade is Silvery Fir Tree, big round plant pot anonPotato Leaf heirloom with very distinct leaf morphology. These cultivars come from a similar region of eastern Europe , and our WGS phylogeny supports a region specific breeding history. The relationships between the Potato Leaf and nonPotato Leaf heirlooms are not well resolved in our WGS-based phylogeny, probably as a result of close relationships between the cultivars and interbreeding. To identify any breeding history specifically related to the Potato Leaf Morph, we performed PHYLONETWORKS analysis using the WGS SNPs . We identified four hybridization events, relating to C mutants . It is noteworthy that Prudens Purple with a unique mutation at the Clocus is not part of this series of hybridization events . These hybridization events suggest a breeding effort for desirable traits associated with this morphology. In addition we also analyzed chromosomes 1, 6 and 12 and found unique hybridizations for all of these chromosomes . These data suggest that analyzing a much larger group of tomato cultivars for hybridization history could be very fruitful.When doing large-scale field studies, it is difficult to understand how all the collected data points relate to each other, and what the causative relationships are . We performed several key correlation tests between measured traits , but to test all traits we would need to perform 91 independent correlation tests. As such, to decipher how all the physiological and morphological traits measured related to each other, we performed PLS-PM using SMARTPLS3.0 , which gives weighted causative paths with bootstrapping for con- fidence and significance values. In PLS-PM, each LV is a composite value of its associated MVs and forms an outer model . The inner model consists of the connections between LVs, with R2 values indicating the degree to which each endogenous LV is described by the connections to it . Here, the only exogenous LV is leaf shape, as it has only its associated MVs and is descriptive of other LVs. Some LVs are described by other LVs within the model . When the value of a causative LV increases, the corresponding connected LVs change in accordance with their relationship with the causative variable. Similarly, in the outer model, changes in MVs reflect a change in their LV, and thus connect the outer model with MVs to the inner model of LVs. For instance, the MV PC3 has a negative correlation with the LV leaf shape , so that as the value of PC3 decreases, it reflects as a corresponding increase in LV leaf shape . This change is represented as an increase in the roundness of the leaf. This then corresponds to a positive change in yield , which is in turn a reflection of fruit biomass . The model indicates that photosynthesis has a strong positive influence on both fruit BRIX and vegetative biomass but has a negative impact on fruit yield. As photosynthetic rates increase , fruit BRIX increases, but at the sacrifice of yield, an inverse relationship which has long been known . Leaf shape has a negative relationship with vegetative biomass, which corresponds to the decreased leaf complexity with the Potato Leaf Morph . However, leaf shape has a strong positive influence on both fruit BRIX and yield , suggesting that leaf shape influences fruit quality as seen previously by Chitwood et al. . The effect of leaf shape on fruit quality does not work through leaf sugar, as this correlation was not significant. Our leaf sugar measurements were completed in the glasshouse, owing to the complexity of the chemical analyses required, and as such the model was tested without leaf sugar. While our work does not implicitly study mechanisms, the negative relationship between leaf sugar and fruit BRIX is of interest, and may provide some avenues for future research into the mechanisms underlying impact of leaf shapes on fruit quality in tomato. Fig. 6 displays the effect of each trait on the overall output of the plants . Leaf shape has no strong contribution to vegetative biomass.