Although rare events of dispersal to distant islands would probably lead to population divergence and eventual speciation , population genetic data suggested that most species with FFs readily overcome oceanic barriers and show weak population genetic structure across islands . Extensive gene flow across islands associated with FFs suggests that this fruit type represents a trait favouring species cohesion in fragmented landscapes . Our conclusions drawn from population genetic data seem to be at odds with studies on Hawaiian lineages for which high species diversity has been linked to limited dispersal of FFs . These contradictory results are most probably explained by the different habitats in which Hawaiian vs. Galápagos and Canary Island lineages of FF species evolved. According to the previous studies, Hawaiian FF lineages that have undergone extensive diversification mostly occur in moist forests, where limited dispersal by sedentary birds, in some instances because of increased seed or fruit size , could have promoted speciation . Hawaiian tropical moist forests currently cover an area of 6700 km2 , which is substantially larger than the estimated potential area for densely forested zones in the drier Canary Islands and Galápagos , especially if we take into consideration the fact that human impact has significantly reduced the original area of moist forests in Hawai‘i. Thus, high species number as a consequence of limited bird dispersal in forested areas is a more plausible explanation for Hawaiian lineages than for the other archipelagos. In addition to the high availability of forested areas on the Hawaiian Islands, growing raspberries in pots other factors may account for the substantial proportion of ‘woodyFF’ ancestors inferred for this archipelago .

For instance, trait evolution on other Pacific islands prior to the colonization of Hawai‘i may have promoted the acquisition of a woody habit under insular conditions, as suggested for Tetramolopium Ness . In summary, differences in patterns of character evolution and speciation among archipelagos can be attributed to context-dependent conditions for lineage diversification , although common trends across archipelagos also occur .Phylogenetic studies have demonstrated that oceanic archipelagos have promoted the in situ diversificationof many plant lineages. In this sense, oceanic islands could also represent a framework in which to address questions about the opposite pattern: i.e. what factors are responsible for a lack of speciation in certain lineages? Excluding the Hawaiian lineages mentioned previously, our analyses suggest that FFs could be one intrinsic factor involved in species cohesion. FFs are generally linked to high colonization ability and extensive gene flow among islands , which may, at least in part, explain why this type of fruit is often displayed by monotypic lineages . Nevertheless, lack of speciation in extant oceanic lineages is obviously not limited to FF species. DF species also represent a substantial proportion of monotypic lineages . Several factors, most also applicable to FF, monotypic lineages, may account for this fact. Because the colonization of oceanic archipelagos could have been constrained to discrete temporal windows , some of these lineages may have found opportunities for colonization only in recent times, so that there has not been sufficient time for recurrent speciation. In turn, it is also plausible that earlier colonizers had more opportunities for in situ diversification. In line with this argument, niche pre-emption by earlier colonizers may have hindered adaptation to novel environments , limiting opportunities for speciation.

Such a possibility could be further tested by the analysis of habitat differentiation among populations of monotypic lineages with DFs showing widespread distributions . Another complementary explanation is that some DFs are frequently dispersed over large distances , and thus recurrent gene flow among populations would be expected to reduce the likelihood of speciation, as discussed previously for FFs. Molecular studies on Phylica arborea Thouars, for example, indicate that this tree species overcame distances of up to 8000 km during the colonization of multiple oceanic islands around southern Africa despite its DF condition . A final point for consideration is that the actual number of monotypic lineages might be conditioned by taxonomic uncertainty in those plant groups in which limited taxonomic research or cryptic speciation obscures the real pattern of diversification. Further interdisciplinary research, including taxonomic, molecular and ecological data, is needed to identify the most important factors related to lack of speciation in monotypic lineages. Lastly, although poorly studied on islands, other intrinsic factors affecting patterns of diversification include those characters related to habitat colonization and population persistence that are induced by environmental cues . For instance, the architectural organization of trees and shrubs favours phenotypic plasticity across canopy layers, allowing persistence and reproduction even under stressful conditions . In addition, resprouting ability is thought to be another key trait for population persistence . Recent ecological studies on widespread island taxa, such as Pinus canariensis C.Sm. ex DC. , Olea cerasiformis Rivas-Mart. & del Arco and Croton scouleri Hook.f. , have indicated that phenotypic plasticity and resprouting ability play a significant role in habitat colonization across broad environmental gradients in Macaronesia and Galá- pagos. These studies support the idea that not only fruit dispersal traits, but also specialized life history strategies involved in population establishment and persistence, may be important in the successful colonization of remote areas and subsequent patterns of diversification of colonizing lineages .

Further studies using a plant trait perspective may help us to understand the implications of intrinsic factors for lineage diversification.Although the present study suffers from typical constraints of meta-analyses , it constitutes, to our knowledge, the first attempt to summarize the inferences drawn from molecular studies on three paradigmatic oceanic archipelagos. Despite clear differences in physiography and geographical isolation, our analyses revealed some similar patterns among these island systems. Thus, phylogenetic reconstructions of character states suggest that ancestors of species-rich lineages in these archipelagos were predominantly herbaceous and with DFs. Island environmental conditions apparently selected for a shift from an herbaceous to a woody habit in most lineages. In contrast, fruit type appears to be strongly phylogenetically constrained, which may have promoted speciation in DF lineages because of generally limited dispersal ability compared with FF lineages. The complexity of the evolutionary processes and biotas considered here, including diversity in fruit morphologies relevant to dispersal ability and dispersal vectors, places limits on the applicability of these patterns. For instance, our analyses suggest that the relationship between high species diversity and FFs indicated by previous studies could be a particular outcome for plant groups that evolved in moist forests, such as those found in Hawai‘i. Traits related to frequent dispersal , in combination with those favouring population persistence , probably play a signifi- cant role in species cohesion and thus contribute to prevent speciation in some lineages. Although further phylogenetic research with new molecular markers is resolving complex evolutionary patterns in radiating lineages , ecological and population genetic studies on islands, particularly those focusing on monotypic lineages, are still critically needed to allow a broader picture to be developed of how evolution works on oceanic archipelagos.In recent years, plant pot with drainage sustainable irrigation has become a crucial aspect of orchard management to reduce inputs in agricultural systems. In the current global warming and desertification scenario, both environmentally and economically oriented reasons provide the basis for a water saving approach, which has become paramount in irrigated orchards. Automated irrigation management is evenmore important in high-density systems in which growers tend to increase orchard productivity and reduce management costs by mechanizing operations. In the past, irrigation management was commonly based on soil water status or environmental indices. However, tree water status provides the most precise drought stress indices, in spite of soil and environmental conditions. Indeed, plants represent the intermediate component of the soil plant-atmosphere continuum, and their physiological responses are the result of an integration of both soil and environment. This implies an advantage of plant-based over soil-based methods for an accurate irrigation scheduling . Plant water requirements differ among species and even cultivars, making irrigation scheduling and management a complex task for growers. The physiological responses of plants to decreasing water availability are various and depend on evolutionary adaptation and acclimation to new climatic conditions. The olive species has a very wide genetic pool, and includes genotypes that can respond to drought using different mechanisms of leaf dehydration tolerance and leaf morphological and structural adaptations .

Gucci et al. and Lo Bianco and Scalisi found different leaf stomatal regulation among olive cultivars. Connor suggested that olive genotypes use avoidance and tolerance mechanisms under drought. Midday stem water potential is considered a very sensitive parameter of plant water status for irrigation management . However, 9 stem is mostly measured by the Scholander pressure chamber, which does not allow for continuous monitoring and automated irrigation. Recently, plant-based sensing technologies are taking hold for the continuous plant water status monitoring in fruit trees. In most of cases, sensors are mounted on above ground organs such as stem, fruit and leaves . In olive, trunk dendrometers have been associated with tree water stress thresholds and proposed for irrigation management due to their relatively easy installation and stability across the season . In the last few years, emphasis has been given to the use of leaf patch clamp pressure probes for the continuous assessment of olive leaf water status . The output of LPCP probes is expressed as attenuated pressure of leaf patches , which is inversely related to cell turgor pressure . Therefore, the highest values of pp occur around solar noon, as that is the moment in which leaf cell turgor is the lowest. Ben-Gal et al. found an inversion of the pp curve in severely drought stressed olive trees. Thereafter, Fernández et al. classified water deficit states based on the degree of inversion of the curve. State I represented no drought stress and leaves with a non-inverted curve, state II grouped leaves experiencing partial inversion of the curve and mild water deficit, and state III enclosed all leaves experiencing severe water deficit and full inversion of the curve. Fruit-based probes based on linear variable displacement transducers can provide good information on fruit growth, which on a diel scale is mostly dominated by water in- and out-flows, rather than carbon gain; thus, fruit diameter variations respond to water deficit . Fernandes et al. studied olive FD dynamics in response to water deficit, suggesting the appropriateness of fruit gauges for continuous plant water status monitoring. Although FD and pp are strictly related to soil water availability and plant water status, they are also influenced by environmental variables, crop load, genetic factors and phenology. The derived values of FD and pp represent good indicators of the rate at which water enters and exits leaf or fruit, respectively. The reasoning behind this assumption resides in the fact that they both represents rates of changes – pressure and diameter, respectively – from an initial state, which, in the short term are mainly driven by tissue water exchanges. Plants modulate water movements to and from the two main transpiring organs using several strategies such as osmotic adjustments, stomatal closure or cell-wall elasticity regulation. As a result, we hypothesized that either leaf or fruit water status might be privileged under increasing water deficit in olive genotypes with different drought tolerance/avoidance mechanisms. This work aimed to study olive fruit and leaf water dynamics in relation to tree water status. Our hypothesis was that, similarly to what found in nectarine , the combination of RR fruit and RRleaf might provide an even more accurate identification of plant water status, rather than monitoring each parameter independently. In addition, this study aimed to identify cultivar-specific RR fruit/RR leaf relationships to determine whether the genotypes under study preserve leaf or fruit water exchanges under increasing water deficit, as sink power for water might differ among genotypes.After removing the three-day buffer period from FD, pp, RRfruit, and RRleaf data, a 5-day interval in stage II was obtained . Trends of FD did not highlight different fruit growth dynamics among irrigation levels in stage II, for both NB and MN. FI induced sharper fruit shrinkage than deficit irrigation at 222 and 223 DOY, as fruit with an optimal water status are likely to exchange more water in the warmest hours of the day.

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, 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, drainage collection pot 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. Especially prominent in F. acuminatum and R. stolonifer were proteins with similarity to subtilisin-like proteases. This family of enzymes is mostly associated with plants and particularly plant defense, but subtilisin-like proteases involved in pathogenicity have been described for fungi as well . Since these inhibitors possess sequence similarity to the proteases themselves, the enzymes identified in F. acuminatum and R. stolonifer may be inhibitors, proteases, or a mixture of both. Additionally, proteases can help with host tissue decomposition by breaking down cell wall structural proteins or can serve in degradation of proteins to provide a source of nutrition for fungal growth . For example, the saprotrophic fungal species Verticillium albo-atrum and V. dahliae were described to secrete proteases to break down structural proteins that stabilize the plant cell walls . High proteolytic activity resulting in the degradation of proteins into free amino acids was also reported during fermentation of tempeh by several Rhizopus species . Botrytis cinerea, F. acuminatum, and R. stolonifer also make use of a variety of CAZymes during interactions with the host. Several CAZyme families are involved in the breakdown of physical barriers present in the host tissues, namely the various cell wall components , cell wall reinforcements , and the waxy fruit cuticle. Many of these enzymes, such as polygalacturonases, pectin methylesterases, pectate lyases, and endo-β-1,4-glucanases, square plastic pot mirror the activities of host enzymes active during the ripening-related softening of the fruit . Others, such as cellulases, cutinases, and lipases, degrade components that are not typically degraded during ripening. Production of cellulases is also coupled with enzymes involved in degradation of cellobiose, the disaccharide product of cellulose breakdown. Both B. cinerea and F. acuminatum appear to focus on production of these latter CAZyme families in MG fruit more than in RR fruit. This may be due to the greater strength and integrity of the cell wall in MG fruit, which requires the fungus to mount a larger attack on the physical barriers in order to penetrate into the cells. Degradation of pectin is a hallmark feature of B. cinerea infection of plant tissues .

The principal enzymes responsible for this process are polygalacturonases , pectin methylesterases , and pectate lyases . Both PGs and PLs cleave the α- 1,4-linkages in the homogalacturonan backbone of pectins. PMEs catalyze the removal of methylester groups on the C6 carbons of galacturonan, which allows for further degradation by PGs. Although overexpression of PME inhibitors in Arabidopsis leaves has been shown to increase resistance to B. cinerea , mutations in Bcpme1 and Bcpme2 do not appear to affect virulence in tomato leaves . In B. cinerea, all three classes of enzymes appear to be highly expressed in MG fruit but not as prominently in RR fruit. Not only do the GH28, PL1-7, and PL3-2 families constitute a greater fraction of upregulated CAZymes in MG fruit, but for PGs, PLs, and PMEs that are commonly upregulated in MG and RR fruit, upregulation is consistently greater in MG fruit over RR fruit. Additionally, although no F. acuminatum PGs were detected in MG, the two upregulated PMEs, FacuDN5818c0g1i1 and FacuDN10179c0g1i1, were only active in MG fruit. Moreover, PL1-7 and PL3-2 genes were strongly expressed in MG fruit, with one PL3- 2 gene, FacuDN8473c0g1i1, showing a log2FC of 10.29 at 1 dpi, the highest of any plant CWDE in this treatment. Only one R. stolonifer PG, RstoDN2036c0g1i1, was detected in MG fruit. However, given that this single R. stolonifer PG was one of only two CAZymes found in 1 dpi MG fruit, it is reasonable to believe PG activity in R. stolonifer isbeing underestimated due to low sequence coverage of fungal transcripts in this treatment. The absence of upregulation of any R. stolonifer pectate lyases in any fruit further underscores this point. Given the prominence of pectin degradation in B. cinerea and F. acuminatum, a more targeted analysis of R. stolonifer pectin degradation, especially in MG fruit, is warranted. Degradation of the host cell wall in MG fruit by pathogen enzymes may accelerate ripening and in turn facilitate a more favorable environment for colonization. Pectinderived oligosaccharides have been shown to induce ethylene production in tomato fruit , which further upregulates expression of host CWDEs, including PG. B. cinerea can synthesize its own ethylene via the α-keto-γ-methylthiobutyric acid pathway , though it is still unknown whether the pathogen produces ethylene during interactions with the fruit. Ethylene production during plant infection has also been reported via the KMBA pathway for species of Fusarium , but not, to our knowledge, for R. stolonifer. However, the specific genes involved in the KMBA pathway in B. cinerea or Fusarium spp. have yet to be elucidated. As colonization proceeds, sugar substrates become available due to degradation of cell wall polysaccharides as well as increased access to stored sugars in the fruit. As a consequence, fungi actively infecting RR tomato fruit induced enzymes that metabolize simple sugars. Sugar metabolism is accompanied by expression of CAZyme families involved in the production and modification of chitin, the structural component of fungal cell walls. Chitin production is known to be a hallmark of growth for fungal pathogens . Interestingly, chitin production and modification appear to be prominent not only in RR fruit for each pathogen, but also in MG fruit inoculated with F. acuminatum, where a much greater amount of mycelia growth was observed compared to the other two pathogens. The equal representation of CE4 enzymes in MG and RR fruit inoculated with F. acuminatum is reflective of the ability of this fungus of producing hyphae at either fruit ripening stage. The abundance of polysaccharide-building glycosyltransferases in RR infections with R. stolonifer is also likely connected to the abundant mycelial growth. Other CAZyme families represent more specialized roles in the infection process. Production of enzymes in the AA7 family may be related to the production of polyketide toxins in B. cinerea and R. stolonifer. B. cinerea is known to produce botcinic acid, a polyketide mycotoxin, during infection . However, the AA7 genes detected to be upregulated in fruit infection here are not known members of the botcinic acid pathway, suggesting that B. cinerea may produce additional uncharacterized polyketide mycotoxins during fruit infection. Even though upregulated F. acuminatum genes involved in toxin production are not annotated as members of the AA7 family, fumonisins are products of polyketide metabolism . The observed upregulation of fumonisin biosynthesis related genes indicates that F. acuminatum also produces polyketide mycotoxins during infection of unripe and ripe tomato fruit.

For wind dispersal, modelling suggests that even moderate topographic variability can have large impacts on variation in dispersal distances and directionality . Finally, slope steepness influences dispersal distances of heavy seeds, which are more likely to roll down downhill , and the likelihood of seed dispersal via runoff . Individual plants might also differ in the quantity and quality of animal-mediated seed dispersal due to the actions of thirdparty players. Some of these effects are mediated by vegetation structure, with plants in more open and risky places receiving fewer visits by dispersers . In other situations, animals respond to olfactory, visual or acoustic predator cues, leading to reduced seed removal rates in frugivorous birds , bats and granivorous rodents . In addition, rodents are sensitive to ungulate presence because of trampling risk or disturbance by rooting ; in Q. ilex, the presence of ungulates was associated with lower quality seed dispersal by rodents and changes in caching sites . Finally, responses to predators and competitors can interact with other traits, such as the presence and concentration of deterrents . Insects frequently infest fruit pulp, seeds or dispersal structures, which can affect seed dispersal. Howler monkeys preferentially feed in Ocotea diospyrifolia trees with high fruit infestation by curculionids and low fruit infestation by moths . The seed parasitoid wasp Macrodasyceras hirsutum reduces attractiveness of Ilex integra berries to frugivorous birds through ‘colour manipulation’; infested fruits are less likely to ripen and turn red, decreasing the risk that the fruits will be eaten and wasps killed .

In synzoochorous dispersal, seed infestation can increase the probability of rejection or of immediate consumption , round plastic plant pot but generally reduces caching rates , thus decreasing dispersal quality. However, not all scatter hoarders discriminate between infested and sound seeds, particularly before insect emergence . Note that these synzoochorous examples are based on responses to individual seeds and it is unknown to what degree they translate into selection among trees differing in infestation levels. Insect attack also affects anemochory. For example, Rhinocyllus conicus larvae feeding on C. nutans receptacles induce callus formation, resulting in inhibited seed release, shortened pappus filaments and reduced dispersal distances . In turn, insect infestation is often affected by masting , thereby creating another, indirect pathway through which temporal variation in seed output can affect seed dispersal.Most of what we know about intraspecific variation in seed dispersal represents a snapshot in time—a frame or two in a potentially long movie of life. While these frames might accurately represent the fitness outcomes for an annual plant, the majority of plants discussed in this review are long-lived perennials that are interacting with an extremely dynamic world where both intrinsic and extrinsic factors vary through time. Although we are not in a position to evaluate the overall consequences of this variation, it is important to acknowledge the variation exists. Fruit crop sizes vary between years. Sometimes this variation is relatively subtle and driven by such factors as resource availability or climatic conditions . Sometimes the variation is extreme, as seen in masting species . Different dispersal kernels are necessary to capture mast versus non-mast years , with potentially greater LDD when acorn density is low . The fitness impacts of this variation should depend at least partially on how synchronous fruit crop size variation is in the population and community. Fruit crop size also varies over longer, ontogenetic time scales; crop sizes increase with perennial plant age and size, often plateauing at some point and remaining relatively constant until death, sometimes showing declines with senescence late in life .

Many other intrinsic traits relevant to intraspecific variation in dispersal are temporally dynamic. Fruit/seed size, and most likely such traits as pulp:seed ratio, vary across years . Plant height increases ontogenetically . Rewards and deterrents can change from year to year and in some cases even seasonally. Temporal variation in extrinsic factors, or the ecological context, is perhaps even more extreme. Fruiting neighbourhoods can change from year to year as different individuals and species respond differently to changing resources and climate . Other aspects of habitat structure around individual plants can change through time due to successional processes and demographic processes , as well as anthropogenic impacts . Lastly, interactions with non-disperser animal communities can vary greatly from year to year as a function of, among other drivers, changes in individual crop sizes and in fruiting neighbourhoods, and population fluctuations of other interacting animal species .Although many exceptions exist, much work on intraspecific variation in seed dispersal has taken a more or less univariate approach; for example, the impact of fruit crop size, fruit size or plant height on dispersal. Alternatively, some address multiple traits affecting dispersal and quantify the relative importance of each and the presence or absence of interactions. In a recent study using an individual-plant-based network analysis of frugivory, locations of individual H. succosa trees within the network were determined by a combination of plant height, fruit size and sugar concentration, with shorter individuals with larger fruits and intermediate sugar concentration being most central . Nonetheless, the true complexity of dispersal is often overlooked. In this review we have also taken primarily a univariate approach, which we argue has value, especially at our early stage of understanding the drivers of inter individual variation in seed dispersal. However, it is critical to understand that we do not believe that this is really how the world exists.

We noted the difficulties of knowing what animal seed dispersers base their harvesting decisions on when so many potentially important traits co-vary: fruit size, absolute and relative quantity of reward, seed number and size, nutrients, toxins and more . For example, do frugivores select fruits to harvest based on size per se or on the underlying variation in pulp:seed ratio ? Such complexities surely exist in other dispersal systems as well. For example, in anemochorous plants, the size of the dispersal structure increases with seed mass, but generally not sufficiently to maintain a constant wing loading . Co-variation of seed release height and seed terminal velocity , and of abscission force and terminal velocity have also been reported. It is highly likely that co-variation of traits relevant to seed dispersal is as extensive with wind dispersal as with frugivory. Complexity also arises in animal-dispersed species because foraging animals often make foraging decisions hierarchically . For example, foraging frugivores must first select the foraging patch, then choose the individuals to feed in, and then choose which fruits to harvest from that plant. In addition, multiple cues may be used hierarchically at any single stage of this process. For example, experiments with the large fleshy-fruited shrub C. monogyna elegantly demonstrated hierarchical selection by Turdus migratorius of individual trees in which to forage. First, birds preferred trees with larger crop sizes, but if crop sizes were constant, they preferred plants with larger fruits, and, finally, if fruit size was constant, they preferred plants with greater pulp:seed ratios . Understanding variation in seed dispersal is further complicated by the concomitant inter individual variation in seed dispersers, including sexual dimorphism, ontogenetic changes, inter individual variation in specialization and unique animal personalities . For example, our discussion of fruit size variation in M. communis and its effect on fruit availability to different seed dispersers was based on measured intra individual and inter individual variation in fruit diameters but only mean gape width for the dispersal agents . Interpretations could be different if inter individual variation in the seed disperser species was also incorporated. More generally, 25 liter rount pot inter individual variation in plants and dispersers interact and it might be difficult to understand one without understanding the other . Plants almost certainly respond at the individual level to variation in how seed dispersers interact with them; these eco-evolutionary feedbacks mean that intraspecific variation is important in both sides of the interaction, perhaps even intensifying the individual-level variation in both players . Further complexity is likely in particular dispersal systems, such as for example with diplochorous dispersal, where dispersal is accomplished by a sequence of steps that involve different dispersal agents such as primary dispersal by a frugivorous bird and secondary dispersal by a rodent . We predict that, all else being equal, diplochorous dispersal systems would have even greater inter individual variation in seed dispersal success than non-diplochorous systems given that variability arising during the second phase of dispersal is building on variability created during the first phase of dispersal.

For example, as discussed previously, intraspecific variation in seed size can affect selection by both frugivorous birds and rodents, sometimes in the same way and sometimes not.While we show substantial evidence that drivers of intraspecific variation in seed dispersal are diverse and pervasive, we also reveal large gaps in our understanding, partly due to a paucity of research directly addressing intraspecific, especially inter individual, variation in seed dispersal, and partly due to the complexity of interactions among drivers. Our understanding is limited further by the existing empirical work’s focus on the quantity of seed dispersal, with much less consideration of the quality of dispersal or LDD. Of particular interest are the intrinsic trait-based drivers that can respond to natural selection. The best-supported and best-understood intrinsic driver of inter individual variation in seed dispersal is crop size; with more seeds produced, more seeds are dispersed. Crop size is also likely the most widespread driver, being relevant to most if not all forms of dispersal. Though less well supported and less well understood, fruit/seed size is likely the second most widespread intrinsic driver. Again, it seems to be relevant to a broad range of seed dispersal modes. However, when it comes to animal-mediated dispersal we do not have a good understanding of the ultimate cause of size-based fruit or seed selection—is it fruit/seed size per se, or some co-varying trait such as pulp:seed ratio? Remaining intrinsic drivers are even more poorly understood, though apparently range from widespread but weak, such as plant height, to sporadic and variable in strength, such as colour polymorphism. For extrinsic drivers, a variety of studies have addressed the impact of fruiting neighbourhoods on inter individual variation in seed dispersal, but we do not understand well when to expect competition for dispersers and when to expect facilitation of dispersal. With respect to habitat structure, much relevant work has been from the perspective of anthropogenic impacts of habitat fragmentation and degradation on seed dispersal rather than from the perspective of interspecific variation in seed dispersal. Beyond limited empirical work, we are further hindered by an even greater lack of theory related to the drivers of intraspecific, especially inter individual variation in seed dispersal. While there have been some theoretical developments around fruit crop size and seed dispersal success , we are aware of no other developed theory that can guide our understanding of the drivers of inter individual variation in dispersal and potential demographic and evolutionary responses to such variation. Looking forward towards potential research directions, in Box 2 we highlight a selection of outstanding questions concerning intrinsic drivers of intraspecific variation in seed dispersal that we personally believe to be especially informative and intriguing to answer. We present these questions as a starting point to advance our understanding of intraspecific drivers of seed dispersal. One promising approach to answer these questions and disentangle the complexity inherent in intraspecific seed dispersal is a frugivore-centred modelling approach . This approach advocates parameterizing field data on intrinsic animal factors and behaviour, as well as extrinsic landscape factors, to test and quantify the strength of the variables affecting the spatially explicit deposition of seeds across the landscape . Mechanistic simulations can be used in a hierarchical manner to test the effect of multiple factors one at a time, to quantify their relative influence on patterns of seed deposition . Studies using this approach have successfully quantified the impact on seed dispersal of edge-following behaviour in a fragmented landscape , fruiting neighbourhoods and drivers of reduced LDD . Although primarily envisioned to study endozoochory, similar methods have been applied to epizoochory and other dispersal modes by considering relevant intrinsic and extrinsic factors . Additionally, a powerful molecular approach that matches individual seeds or seedlings to maternal plants across dispersal modes is also promising for studying individual variation in seed dispersal and may compliment simulation modelling approaches. D

The work presented in this chapter is thus an attempt to narrow down the most likely mechanism that cytokinin uses to affect WUS transcription, translation, and protein distribution. Surprisingly, the results found that elevated levels of cytokinin did not directly affect WUS transcription, nuclear localization, or stability, nor did cytokinin have any significant effect on CLV3, eliminating a possible indirect mechanism. Instead, a novel absence of cytokinin response was identified in the CZ, and evidence suggests that this zone is maintained both by the lack of transcription, and by an unknown repressive mechanism that can affect B-type ARR proteins.Cytokinin responses were also correlated with WUS protein stability, starting roughly 12 hours after exogenous treatment. Auxin however, dramatically reduced WUS protein levels within just 4 hours, suggesting that this hormone has a more direct effect on protein stability. This suggests a model where auxin responses in the CZ and PZ cells stimulate protein degradation pathways that confine WUS proteins to the RM, where cytokinin responses may favor protein stability.In order to identify which part of the WUS-CLV3 feedback loop is affected by cytokinin responses, this work began by crossing the pTCSn1:mGFP5-ER reporter was crossed into wus-1 and clv3-2 mutant backgrounds. In untreated plants, both clv3-2 and WT meristems were found to have strong cytokinin responses in the RM, with a faint fluorescent signal extending deep into the pith and provascular tissue . The wus-1 mutant was similar, though its fluorescent signal did not become faint in the deeper tissue layers, presumably because this mutant meristem did not produce pith or provascular tissue. Treatment with exogenous 6- benzylaminopurine for 24 hours did not change the location of the cytokinin response in WT meristems, or in the meristems of either mutant. Instead, 25 liter plant pot the strength of the fluorescent signal was more than tripled in all three backgrounds, suggesting that endogenous cytokinin response mechanism is able to function over a wide range of concentrations.

The enhanced signal was most easily detected in the weakly fluorescent pith cells, but interestingly, the immature leaves, young anlagen, and the apex of the SAM all failed to produce any fluorescent response at all. In both WT and clv3-2 mutants, the absence of cytokinin responses occurred in a circular patch at the apex of the Central Zone and extended two cell layers deep. In wus-1 homozygousmutants, a similar response-free zone was found to be variable, but was detected in 84% of sectioned meristems, and extended only one cell layer deep . The near-complete lack of cytokinin responses in the CZ was unexpected, though presence of this function indicates a previously unrecognized feature of meristem organization involving two opposite and adjacent cytokinin response fields. As exogenous cytokinin applications were not able to induce pTCSn1:mGFP5-ER expression in the response-free zone to any significant degree, the two-component system pCLV3:GR-LhG4 x p6xOP:ARR1ΔDDK-GR was used to ectopically stimulate cytokinin responses specifically in the CZ tissue. As previously described, the ARR1ΔDDK-GR construct is a modified version of ARABIDOPSIS RESPONSE REGULATOR 1 , which activates cytokinin response genes following exposure to dexamethasone. Plants containing both constructs were then crossed to three different lines containing the fluorescent reporters: pTCSn1:mGFP5-ER, pCLV3:mGFP5-ER, and pWUS:eGFP-WUS. Surprisingly, when observed over a 48 hour time course of dexamethasone treatment, the pTCSn1:mGFP5-ER reporter did not immediately occur in the middle of the CZ as expected. For comparison, the pCLV3:mGFP5-ER reporter uses an identical promoter, indicating that the induced cytokinin response should occur in a pattern similar to the middle row in Figure 3.2. Instead, the pTCSn1:mGFP5-ER signal first appeared at the extreme edges of the peripheral zone, where it progressively appeared in adjacent cells in a centripetal manner, slowly constricting the cytokinin response-free zone until it disappeared between 24 and 48 hours later.

The centripetal pattern was visible in both L1 and L2 cells, though the L2 signal was weaker and lagged behind the L1 by 1-3 cell diameters. By 48 hours, the response-free zone was completely lost, and pTCSn1:mGFP5-ER expression become nearly homogenous throughout the SAM.The pCLV3:mGFP5-ER reporter in contrast, was expressed in the apical pattern as expected for the CLV3 promoter. The fluorescent pattern occurred in conical patch of cells at the apex of the SAM, and extended up to four cells deep. The fluorescence levels were mostly uniform, though the L2 frequently had significantly less expression than the other layers. The expression pattern was already fully formed in the absence of dexamethasone treatment, and remained unchanged through at least 24 hours. The fluorescent pattern became broader in proportion to the size of the meristem at 48 hours, but the longitudinal pattern did not significantly change. Deep inside the SAM tissues however, a faint pCLV3:mGFP5-ER signal could be detected, which produced a central hourglass-shaped column more than 20 cell layers deep. At the beginning of the time course, the pWUS:eGFP-WUS reporter produced a nuclear-localized pattern centered on the RM, with a radial concentration gradient spreading into all adjacent cells as expected. This pattern did not change after 6 hours of dexamethasone treatment, but by 12 hours a subtle increase in the number of cells displaying the pWUS:eGFP-WUS fluorescent reporter was apparent. The number of small meristem-like cells also began to increase over time, accumulating in a rootward direction at a rate directly proportional to the loss of the underlying large pith cells. The pWUS:eGFP-WUS expression pattern followed the downward appearance of the new cells, eventually producing a brightly visible fluorescent column extending more than 20 cells deep.

Elongate voids with no fluorescence were visible on either side, similar to the faint column produced by pCLV3:mGFP5-ER. Interestingly, long term ectopic induction of the pCLV3:GR-LhG4 x p6xOP:ARR1ΔDDK-GR system did not significantly change the volume of the SAM over the first 24 hours, but by 48 hours the SAM volume had quadrupled. This exponential growth pattern continued in plants subjected to prolonged 120 hour treatments, eventually producing a spherically swollen SAMs 1- 2mm in diameter, with frequent superficial cracks . Curiously, while the change in cell proliferation appeared to be abrupt in the pTCSn1:mGFP5-ER and pCLV3:mGFP5-ER reporter backgrounds, the proliferation rate in the pWUS:eGFP-WUS background was more gradual, beginning at least 12 hours earlier than in the other two lines. This precocious behavior may be related to the concentration of WUS proteins in this line, as the presence of the pWUS:eGFP-WUS construct can complement wus-1 mutants, and likely double doubles the concentration of WUS proteins in the presence of the WT copy of WUS.While CLV3 does not appear to induce or respond to cytokinin to any significant extent, WUS proteins displayed a more complicated pattern as shown by pWUS:eGFP-WUS reporter in Figure 3.4: Part of the WUS pattern overlaps with the cytokinin-response-free zone, and typically no WUS was found in either the deep RM or the PZ, where cytokinin responses were clearly present at comparable time points. The failure of WUS to activate cytokinin responses in the CZ is somewhat surprising, as 24 hours of exogenous 6-bap treatment moderately increased pWUS:eGFP-WUS fluorescent levels in both WT and clv3-2 mutant backgrounds . On the other hand, if cytokinin is required to activate WUS transcription, the presence of WUSexpression in tissues that lack a clear cytokinin response is equally difficult to explain. When cytokinin responses are eliminated with the cytokinin receptor triple mutant ahk2/3/4, only trace amounts of pWUS:eGFP-WUS fluorescent signal could be detected in seven day old plants. The lack of cytokinin responses was further confirmed by treating pWUS:eGFP-WUS x ahk2/3/4 plants with exogenous 6-bap, which did not significantly change the fluorescent pattern . However, the ahk2/3/4 mutant was quite variable, as 74% of examined SAM tissues displayed no fluorescence, while the remaining 26% ranged from faint GFP patterns to nearly full WT-like patterns . To deplete native cytokinin in WT meristems without the physical defects of the ahk2/3/4 mutant, the dex-inducible construct p35S:GR-LhG4::p6xOP:CKX3 was used to over-expresses CYTOKININ OXIDASE 3 , which degrades native cytokinin molecules. Following 24 hours of dexamethasone treatment in this background, the pCLV3:mGFP5-ER reporter showed no significant change in expression . Parallel attempts to study pWUS:eGFPWUS in the p35S:GR-LhG4::p6xOP:CKX3 background produced extremely variable results during the first 24 hours, black plastic plant pots ranging from the complete absence of fluorescent signal, to near-WT patterns, but became consistent by 48 hours of dex treatment.When WUS transcription was checked with RT-PCR however, both WT and ahk2/3/4 mutants background were found to have detectable WUS transcription localized to the RM .

The expression pattern of WUS also largely unchanged in ahk2/3/4 mutant RNA in-situ’s, suggesting that cyokinin responses primarily affect WUS protein. Further RNA in-situ’s following the time course treatment of the pCLV3:GR-LhG4 x p6xOP:ARR1ΔDDK-GR system found that cytokinin did not significantly increase WUS transcription in the CZ cells .This indicates that the pWUS:eGFP-WUS fluorescence observed in CZ cells is a product of protein movement, not local transcription. The RNA in-situ’s further revealed that WUS transcription patterns also expanded in a rootward direction, similar to the pWUS:EGFP-WUS pattern shown in Figure 3.2. By 48 hours, WUS expression was clearly found throughout the entire volume of the enlarged RM, with the exception of L1 and L2, which had little or no WUS transcripts. In many cases, large elliptical voids appeared in the peripheral zone, which corresponded to the presence of lateral anlagen. When two voids were present simultaneously , the central RNA expression pattern is reminiscent of the central column displayed by the pWUS:eGFP-WUS reporter in Figure 3.2. Though WUS is known to be non-cell autonomous, the close correlation between RNA and GFP patterns suggests that WUS proteins has a short mobile range, here estimated at 3 cell diameters.Previous research has shown that the non-cell autonomous movement of WUS protein does not have tissue-specific patterns , suggesting that the plasmodesmata are unlikely targets of cytokinin regulation. In order to explore other possible means of protein movement regulation, this study began by performing hand-cut longitudinal sections of pWUS:eGFP-WUS plants, providing an un-biased view of the WUS concentration profile in the deeper layers of the SAM, thereby avoiding the loss of signal associated with tissue depth. Special care was taken to avoid saturating the pWUS:eGFP-WUS reporter during imaging, so that semi-quantitative analysis might reveal subtle patterns . In untreated plants, the pWUS:eGFP-WUS reporter revealed a nearly symmetrical concentration profile, with a triangular peak centered on the RM, tapering off over 3-4 cell diameters . The location of the peak varied between L3-L5 in different sections, which likely reflects error introduced by tangential or oblique cuts. Above the peak, the fluorescent gradient was strongly linear, tapering to near undetectable levels in L1 cells. In the deep meristem tissues, the rootward gradient was equally linear and symmetric for the first 2-3 cell diameters, but then began to flatten out into a low but relatively constant background signal. It is not clear how much of the deep-layer signal reflects the presence of WUS, as the pWUS-eGFP-WUS reporter did not display its characteristic nuclear-localized pattern in these cells. Instead, the fluorescent signal largely co-localized with the developing chloroplasts in deepest cells layers, suggesting that this background signal is at least partially derived from chlorophyll auto-fluorescent noise. However, no such noise can be detected in the absence of pWUS-eGFP-WUS, or when histone or ERtagged fluorescent proteins are used , strongly implying that this background signal reflects the actual WUS protein distribution. When pWUS:eGFP-WUS plants were treated with exogenous 6-bap for 48 hours, no significant changes were observed in the upper gradient , either in the slope or in the total fluorescent concentration. The signal started to diverge by L4 however, where fluorescent signal became as much as 2x brighter down through at least L10 .When cytokinin responses were ectopically induced in the pCLV3:GR-LhG4 x p6xOP: ARR1ΔDDK-GR background, the pWUS:eGFP-WUS reporter produced patterns very similar to the exogenous cytokinin treatments. The upper gradient remained unchanged, while the deeper cell layers starting at roughly L5 doubled their fluorescent signal. A time-course analysis further revealed that the deep-meristem signal began to appear after 12 hours, and was fully formed by 24 hours. Interestingly, by 48 hours the gradient in L1-L3 cells suddenly increased their fluorescent amplitude by 140%, yet the slope of the gradient in these cells remained unchanged.When cytokinin responses were reduced with the p35S:GR-LhG4::p6xOP:CKX3 construct, a slightly different pattern emerged.

The declining trend began before significant numbers of senescent leaves were observed, and continued unperturbed even after most leaves had been lost In comparison, rosette leaf initiation rates revealed a nearly constant production rate at about 1.1 leaves per day, which is about the same as the initial rate of flower bud production. Meristem growth stopped between 26-28 days after induction, indicated by the simultaneous plateau in both immature green bud numbers and total flower numbers . In many cases the buds around the arrested meristem remained green for several days, during which time they can be induced to resume growth by removing the fruits or following the senescence of the old fruits. Flowers produced during the transition to this quiescent phase were often small, infertile, and had petals that did not exceed the sepals. The visible symptoms of senescence eventually appeared as an abrupt color change, proceeding from pale green, yellow, dark red in less than 48 hours, and eventually became brown as the tissue dried out. Senescence affected multiple tissues within the apical region simultaneously, including the SAM, 1-2 mm of the subtending stem, and all attached flower buds within that region, while tissues immediately below remained green . No abscission layer was detected. One possible trigger for such apical senescence may come from the leaves, which showed an increasing trend of senescence prior to meristem arrest . To study the possibility that apical senescence might be triggered by mobile signal produced by senescent leaves, growing strawberries vertically a further experiment was performed that removed leaf tips from adult-phase leaves before they began to display senescent-related color changes.

Removal was timed to coincide with the maximum leaf length growth rate, well before senescence could occur. The results however, show that this actually had the opposite effect, as meristem arrest occurred on average 4.6 buds, or about 1.8 days sooner in leaf-tipped plants than it did in the controls . Alternatively, the fruits might be the source of a growth inhibitor, as predicted by the alternate bearing inhibitor hypothesis. To eliminate this possibility, the flowers were removed on a daily basis in order to observe changes in meristem activity and the time of growth arrest. Because previous results had shown that the rate of immature bud production and total stem height were closely correlated during the flowering period , internode lengths were measured as a proxy for meristem activity in A. thaliana. The resulting data revealed that both the controls and de-fruited plants displayed a similar declining trend of meristem activity that eventually reached zero growth . However, de-fruited plants clearly offset the time of meristem arrest through an extended period of almost linear bud production rates. This linear trend began a day before the control meristems slowed to zero growth, and lasted foral most a week before resuming the declining trend that the control plants had already completed. To verify the effects of fruit load in Avocados, SAM tissue was collected from branches with and without subtending fruit, four months after anthesis. To test the dominance and inhibitor hypothesis, the expression profile of four genes were measured with qPCR, yet this analysis found no significant patterns related to the presence of the fruit .In the carbohydrate competition model, the fruits are thought to consume the majority of available nutrients, leaving little if any for the rest of the plant. The remaining parts must either reduce their growth in direct proportion to the limiting nutrient, or trigger starvation responses in order to survive short-term depletions.

In most plants, the two most commonly encountered nutrient depletions involve carbohydrates and nitrogen, both of which have well-characterized response patterns that also show a significant degree of overlap. Under carbohydrate starvation conditions, for example, the plant tissues typically suppress respiration and growth while consuming their starch reserves. Eventually both proteins and lipids are degraded, releasing free amino acids and nitrogen in the form of ammonia. Some nitrogen is recovered in glutamine or asparagine biosynthesis, while the rest either diffuses into the media or is consumed by the nearest available sink tissue. Under prolonged conditions, large portions of the cytoplasm are consumed by autophagy, in which even organelles are degraded in lytic vacuoles containing cysteine/serine proteases. These vacuoles eventually coalesce until the cell consists of little but the nucleus and a large vacuole, which is followed by cell death under extreme cases. This pattern of responses is also strongly reflected in the present study. Starch breakdown is predicted both by trehalose signaling and direct digestion by AMY1. Although an increase in asparagines biosynthesis was not detected, the methionine gamma-lyase enzyme is known to release ammonia, and two nitrate transporters were upregulated, perhaps reflecting a decrease in free nitrates. Autophagy is consistent with the upregulation of an array of cystein/serine proteases, the near-lack of DNA laddering, and the presence of MC3. Carbohydrate starvation has also been reported to reduce osmolarity and membrane permeability, which parallels the observation of a weak plasmembrane, and the up-regulation of desiccation responses found by the present study.

Nitrogen starvation in contrast, is associated with the suppression of most chloroplast and photosynthesis related enzymes, loss of starch reserves, and the increase in uptake transporters and various storage compounds, including asparagine, glutamine, and various organic acids, while reducing losses that occur through degradation. Prolonged shortages typically result in anthocyanin production. Autophagy also seems to be an important part of nitrogen recycling in senescent leaves, though this may not have been detected in published studies that rely on short term starvation experiments.In the present data, the symptoms of nitrogen starvation are equivocal. Two photosystem subunits were reduced , which is broadly consisted with the degradation of the chloroplasts predicted by FZL and ELIP2. Autophagy signals do occur in the data, but the collected tissues were not obviously senescent, suggesting that autophagy may be more closely related to carbohydrate starvation. The increase of PAP1 is consistent with the biosynthesis of flavonols and other red pigments under nitrogen stress, though the much stronger increases in GL3, PAP2, and MYB12 reported by were not detected in the present study. The nitrate importer NRT1.2 was increased, but this conflicts with the earlier interpretation of amino acid catabolism in the present data. Thus the IM does not appear to be synthesizing storage proteins as predicted by nitrogen starvation studies. In consideration that the growth conditions used in this study included supplemental nitrogen fertilizer, the existence of a nitrogen shortage is unlikely. Instead, the relatively weak nitrogen starvation signal found here is potentially a consequence of the much more significant carbohydrate starvation response.Among the candidate induction genes that might be inhibited by high fruit loads, the results found that none of the floral induction pathways were reduced. Instead the expression of FT was strongly increased , as were several downstream targets including SOC1, AGL71, and perhaps also AGL44. One possible explanation for this pattern might be found in the expression pattern of FT, which based on microarray data is also produced by the ovaries and immature fruits, in addition to the leaf and stem vasculature. The failure to remove all immature flower buds during tissue collection might then bias the results in favor of these induction genes. In avocado trees, the TFL1 and DAM1 paralogs showed no significant response to fruit load . The bio-synthetic enzymes that produce phloridzen are currently unknown, so the presence of this compound cannot be evaluated with the present data.The fruit dominance hypothesis also does not seem to be strongly supported by the present study, drainage planter pot as all detected parts of the auxin response pathway were suppressed by high fruit load conditions. However, Arabidopsis is among the minority of species that do not have strong apical dominance responses, which could imply that this finding is an artifact. The failure to abscise immature fruits like many alternate bearing trees, for example, may indicate the lack of a functional fruit dominance system. However, the observation that isolated nodes of Arabidopsis can suppress axillary bud growth following auxin treatment , and that the lower portion of Arabidopsis branches initiate growth in a basipetal pattern, suggest that the dominance mechanism has not been lost, but is instead suppressed. A likely candidate for this suppression is the plant hormone strigolactone, which is able to suppress branching in the shoot when expressed in the roots.

In A. thaliana, the widespread expression of the strigolactone biosynthesis gene MAX1 in the vasculature is consistent with the broad suppression of dominance pathways in the species. Given that strigolactones appear to inhibit bud growth by blocking their ability to export auxin, this system has interesting parallels with the 2nd fruit drop, king fruit dominance, and even the number of flowers produced by axillary inflorescences. Immature fruits are already known to export auxin, suggesting that the plant may control their numbers by secreting root or shoot derived strigolactone into the inflorescence. The up regulation of strigolactone production under starvation conditions is certainly consistent with the increase in MAX1 expression levels found in the present microarray data. Such a role may explain the different alternate bearing amplitudes that were found when avocado trees were grafted to different rootstocks. Expression of ARF2 and RMS2 paralogs in Avocado trees however, did not reveal a significant response to fruit load .Another potential candidate for fruit inhibition might also include the trehalose signaling pathway. Although the precise role of this signaling sugar is not well understood, biosynthesis mutant are lethal, and high concentrations of trehalose-6-phosphate do seem to be correlated with starch biosynthesis. Accumulating evidence however, suggests that trehalose may have a central role in controlling the broad details of cellular metabolism. Several TPP genes are upregulated in response to nitrate treatments , and also by carbohydrate starvation. This mirrors the findings of the two trehalose phosphate phosphatases that were identified in this study, and in a recent profiling experiments with alternate bearing trees. Trehalose has also been shown to inhibit the kinase activity AKIN10/11, which has broad effects throughout the cell, and may explain how trehalose can regulate pathways such as cell size and stress tolerance. Intriguingly, the vegetative-adult phase change has also been implicated to be a product of carbohydrate supply, which also involves trehalose signaling.Although the data supports the existence of a massive senescence response, much of this signal disappears when the symptoms of carbohydrate starvation are considered. This is consistent with observations of the tissue during collection, which was often still green and displayed no external symptoms of senescence. The only remaining sign of biologic stress is the slight increase of AtRLP54 expression levels.The inflorescence meristem of Arabidopsis is a determinate growth, known to stop functioning after producing a predictable number of flowers in the Ler ecotypes. Under the growth conditions used by this study for the Col-0 ecotypes, the time of meristem arrest was slightlymore variable, but otherwise displayed a nearly identical pattern. Growth arrested meristems however, do not immediately terminate their activities, but instead exists in a quiescent state for several days, during which they can resume growth when the subtending fruits are removed. The present data suggests that this behavior is driven to a large extent by the re-allocation of carbohydrate resources. Interpreted in this way, the quiescent state is comparable to the survival phase exhibited by excised maize root tips, where growth could be resumed by adding supplemental sugar to the media. However, starvation isn’t sufficient to explain all of the behaviors of the inflorescence meristem. In contrast to previous report of a linear rate of anthesis, a closer examination of meristem activities revealed a number of subtle trends. When measured in terms of anlagen/day, the vegetative meristem is found to have a nearly constant rate of production that does not change with the juvenile/adult phase transition, which occurred between leaves 6-8 . After induction, the inflorescence meristem revealed a rapid rise in flower buds/day, a period of time that corresponds to an enlarged SAM diameter, and an increase in gibberellic acid biosynthesis. As anlagen production requires a finite surface area in order to develop, the increase rate of bud production may be related to the larger diameter of the meristem during this time.

The differences in fungal spore concentration were adjusted to ensure uniform and comparable development of lesions in tomato fruit. In the case of R. stolonifer inoculations of MG fruit, we also tested a concentration of 1,000 spores/µl but no differences in fruit responses or fungal growth between this concentration and 30 spores/µl were observed. Inoculated fruit were incubated at RT in high humidity chambers. For mock inoculations, the same procedure was followed but without the addition of the inoculum. The pericarp and epidermis of the blossom end were collected at 1 and 3 days post-inoculation , immediately frozen in liquid nitrogen, and stored at −80◦C until use. One biological replicate consisted on average of eight fruit, and five biological replicates per treatment were obtained.Tomato fruit tissues were ground using a Retsch R Mixer Mill MM 400 and RNA was extracted from 1 g of fine-powdered tissue according to the procedure described in Blanco-Ulate et al. . Fungal RNA from the in vitro cultures was extracted using TRIzol and purified using the Quick-RNA MiniPrep Kit following the procedure described in MoralesCruz et al. . The RNA concentration and purity were assessed with the Qubit 3 and the NanoDrop One Spectrophotometer , respectively. Gel electrophoresis was used to confirm the RNA was not degraded. Barcoded cDNA libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit v2 . Quality control of the cDNA libraries was performed with the High Sensitivity DNA Analysis Kit in the Agilent 2100 Bioanalyzer . 50-bp single-end libraries were sequenced on the Illumina HiSeq 4000 platform in the DNA Technologies Core of the UC Davis Genome Center. In total, 18 libraries were sequenced for B. cinerea , 17 libraries were sequenced for F. acuminatum , and 17 libraries for R. stolonifer .

Quality trimming of the raw reads was performed with sickle v1.331 and adapter sequences were removed with scythe v0.9912 . The basic quality measurements were assessed with FastQC3 . To generate transcriptome assemblies for F. acuminatum and R. stolonifer, stacking flower pot tower reads from samples infected with each of these pathogens were mapped to the tomato genome using STAR 2.6 . Reads that failed to map to the tomato gene were pooled with the reads from the in vitro samples and used for de novo transcriptome assembly. Transcriptomes were assembled with Trinity 2.5.1 using default parameters . Quality of the assemblies was assessed with BUSCO 3.0.2 using the “fungi odb9” dataset, while basic assembly metrics were obtained with Transrate 1.0.3 . Potential contaminanttranscripts were identified via BLAST using both the blast nr database and the UniProt database. Functional annotations for transcriptomes of all three fungi were created using Trinotate 3.0.1 . The default Trinotate parameters were used to retrieve the best BLAST hits as well as annotations for Gene Ontology terms, Pfam families, Kyoto Encyclopedia of Genes and Genome pathways, EggNOG predictions, and SignalP sequences. Custom BLAST databases were incorporated according to the Trinotate manual for the Transporter Classification Database and the Pathogen-Host Interactions database . Custom HMMER alignment results for HMM profiles from dbCAN7 and fPoxDB8 were incorporated similarly.To determine if F. acuminatum and R. stolonifer show similar patterns of infections in tomato fruit as B. cinerea , we did side-by-side inoculations of fruit at two developmental stages: unripe and ripe . As displayed in Figure 1A, we confirmed that all fungi were unable to cause rotting in MG fruit but aggressively colonized RR fruit. These results were further validated by quantifying fungal biomass based on relative expression of fungal reference genes via qRT-PCR .

At 3 dpi, RR fruit inoculated with B. cinerea and F. acuminatum showed water-soaked lesions of approximately 15 mm covered by dense mycelia, whereas RR fruit inoculated with R. stolonifer were almost decomposed and entirely covered by mycelia. Although no lesions were observed in MG fruit when inoculated with any of the pathogens, some differences in fungal growth and tomato responses were observed. Inoculations with B. cinerea and R. stolonifer did not show any visible mycelia, whereas F. acuminatum inoculations showed limited hyphal growth without disease symptoms. All three fungi induced a necrotic ring surrounding the inoculation sites during the incompatible interaction with MG fruit, yet F. acuminatum inoculations caused dark and wide rings while fruit infected with R. stolonifer developed a weaker response. Because we were not able to visually detect any hyphal growth of B. cinerea and R. stolonifer inMG fruit, we used a microscope to observe whether the spores germinated in the inoculated wounds. At 1 dpi, B. cinerea spores were mainly ungerminated or in the process of germination . By contrast, F. acuminatum and R. stolonifer already showed active hyphal growth, indicating that spores of these fungi germinate earlier on MG fruit. At 3 dpi, some hyphal growth was also observed for B. cinerea. Together, these observations suggest that the incompatibility of the interaction between these fungi and MG tomato fruit occurs after spore germination. To provide initial support that both F. acuminatum and R. stolonifer are capable of inducing disease responses in the host, like B. cinerea, and do not merely behave as saprotrophs , we evaluated the expression of the host gene SlWRKY33 , which is well-known to be pathogen-responsive but is not induced by abiotic stresses . To test that the induction of this gene occurred only as a result of inoculation and not wounding, we included a mock-inoculated control in our analyses. The expression patterns of SlWRKY33 measured by qRT-PCR reflected the accumulation of fungal biomass and the presence of lesions in each of the treatments . At 1 dpi, expression of SlWRKY33 was induced by inoculation with both B. cinerea and F. acuminatum but not with R. stolonifer or mock inoculation in MG fruit. In RR fruit, pathogen-induced SlWRKY33 was detected for all three pathogens at greater levels than found in MG fruit.Our observations of lesion development, fungal biomass, and activation of pathogen responses led to the hypothesis that F. acuminatum and R. stolonifer display a similar necrotrophic behavior in tomato fruit as B. cinerea. Therefore, to discover pathogenicity or virulence factors in these fungi that are important for necrotrophic infections, we performed a genomewide transcriptomic analysis of inoculated fruit at both time points as well as in vitro cultures. Due to the lack of publicly available genomic data for F. acuminatum and R. stolonifer, we assembled de novo transcriptomes for both of these pathogens from our cDNA libraries following the Trinity pipeline . Using the fungal ortholog dataset of the Benchmarking Universal Single-Copy Orthologs tool , we determined that our assemblies presented high completeness, with 88.2 and 90.3% of F. acuminatum and R. stolonifer matches being complete, respectively. Our F. acuminatum transcriptome contained 20,117 unique transcripts, danish trolley while our R. stolonifer transcriptome contained 19,754 . We then used homology-based annotation to obtain information on gene functions for each of the transcriptomes, including the B. cinerea B05.10 ASM83294v1 . We annotated transcripts based on nine separate functional classifications, including GO , Pfam domains , Pathogen-Host Interaction , membrane transporters , Carbohydrate-Active Enzymes , and fungal peroxidases .

Each type of functional annotation was represented by a similar percentage of annotated transcripts across all pathogens . The specialized enzyme classifications of peroxidases and CAZymes made up a relatively small fraction of the annotated transcripts, whereas general functional classifications such as GO, Pfam, and KEGG descriptions were available for at least 70% of the annotated transcripts for all pathogens. Annotations for all three transcriptomes can be found in Supplementary Table S3. Although the F. acuminatum and R. stolonifer transcriptomes are preliminary and may require further curation and validation, we consider that they are a valuable resource to perform gene expression analyses and to shed light on the infection strategies utilized by these fungi.First, we performed principal component analysis to determine if the fungal-inoculated and in vitro samples could be discerned based on the expression of the fungal transcripts. The PCAs revealed that all samples clustered by treatment . In most cases, the first component clearly differentiated the MG fruit from the RR fruit inoculations and the in vitro samples. Then, we determined DEGs between inoculations of MG or RR fruit and in vitro cultures for each pathogen. Across all comparisons, we detected 6,488 B. cinerea DEGs , 6,154 F. acuminatum DEGs , and 8,777 R. stolonifer DEGs . The number of DEGs for R. stolonifer were mainly identified in the RR fruit comparisons, as the low amount of fungal biomass in MG fruit samples did not allow for an in-depth sequencing coverage of the fungal transcripts. To confirm the accuracy of the DEG analysis, we selected a subset of genes for each pathogen to validate their expression using a qRT-PCR approach . Our results confirmed that the gene expression values were consistent, showing significant Pearson correlation coefficients and between the RNA-seq and the qPCR expression data . We further evaluated the fungal DEGs based on whether they were commonly or uniquely expressed under specific treatments, which can provide insight on particular sets of genes that are relevant during incompatible or compatible interactions . For each pathogen, genes uniquely upregulated in RR fruit constituted a sizable fraction of upregulated genes . This result may be influenced by the fact that RR fruit samples had more coverage of fungal transcripts in the RNA-seq experiment than MG fruit samples, which is a technical limitation of this type of study. Nevertheless, the comparisons of common and unique DEGs among treatments for each of the pathogens support the results of the PCAs, indicating that these fungi display a specific behavior in each of the fruit stages at early and late time points after inoculation. We also identified upregulated DEGs shared across categories that are likely to represent core pathogenicity factors during fruit infections.To gain insight into key biological processes that are relevant during compatible or incompatible fruit infections, we performed GO enrichment analyses of the upregulated DEGs in all combinations of ripening stage and dpi for each pathogen . We mainly focused on GO terms of the “biological process” class that were significantly enriched and appeared to be involved in pathogenesis or fungal growth in the host tissues . Upregulated DEGs from all comparisons, except for R. stolonifer MG inoculations, were enriched in oxidation reduction processes . A closer inspection of these DEGs revealed functions that are likely to be involved with pathogenicity, such as catabolism of ROS [e.g., superoxide dismutases , catalases , peroxidases] and breakdown of cell wall molecules such as cellobiose and lignin . In B. cinerea, the SOD BcSOD1 was induced in both MG and RR fruit at 1 and 3 dpi. Additionally, BcSOD3 was upregulated only in MG fruit at 1 dpi, and BcSOD2 is upregulated only at 1 dpi in MG and RR fruit. Although two potential SODs, FacuDN9613c0g1i1 and FacuDN4275c0g1i2, were employed by F. acuminatum in all treatments except 1 dpi MG, none of the seven putative SODs identified in R. stolonifer were upregulated in any of the treatments. To further identify enzymatic scavengers of hydrogen peroxide , we examined the upregulated DEGs of each pathogen which showed significant similarity to members of the Fungal Peroxidase Database. This analysis revealed differences both in the classes of enzymes used in each pathogen and the treatments in which they were used. For example, in B. cinerea, only two known catalases, BcCAT2 and BcCAT4 , were found to be upregulated during tomato fruit interaction. Both of these were only active in MG fruit. In contrast, F. acuminatum exhibited very strong induction of two predicted CATs, FacuDN12367c0g1i1 and FacuDN13048c0g1i1, at 1 dpi in RR fruit but not in MG fruit, although a handful of CATs and catalase-peroxidases were upregulated less strongly across both MG and RR fruit. In all F. acuminatum-inoculated samples, there was also an enrichment of DEGs involved in hydrogen peroxide catabolism , further highlighting the importance of fungal responses to oxidative stress during fruit colonization. In R. stolonifer, peroxidases were only upregulated at 1 dpi in RR fruit and included two 2-cysteine peroxiredoxins , one cytochrome C peroxidase, and one glutathione peroxidase . Additionally, in all B. cinerea-inoculated samples, DEGs annotated with the oxidation-reduction process GO term included enzymes in the biosynthetic pathways for the phytotoxins botrydial and botcinic acid.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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