Category: hydroponic growing

Achenes are small single-seeded fruit, whereas the receptacle is considered to be anatomically equivalent to floral meristem tissue . F. × ananassa is an allo-octoploid that originated as a synthetic hybrid between the octoploid species Fragaria chiloensis and Fragaria virginiana . Strawberry is affected by several pathogens including fungi, bacteria, viruses and nematodes. The most economically impactful pathogens of strawberry are fungi, which can infect all parts of the plant and cause severe damage or death . Amongst the fungal pathogens, the ascomycete Botrytis cinerea is considered the primary pathogen of harvested strawberries in the world leading to impactful economical losses to the strawberry industry. B. cinerea causes grey mould in fruit and senescing organs but can also affect vegetative tissues . Under wet conditions, more than 80% of strawberry flowers and fruits can be lost if plants are not sprayed with fungicides .B. cinerea has no apparent host specificity and can infect more than 1000 plant species . The pathogen is found worldwide and causes disease in many fruit, flower and leafy vegetable crops . B. cinerea is classified as a necrotroph, meaning that it prefers to infect and grow on damaged or senescing tissues, eventually causing tissue death. The inoculum of the fungus is highly abundant and ubiquitous and usually comes from infected plant tissues . B. cinerea mainly enters the host via wounds or natural openings . Infections of non-senescing or unripe plant organs usually lead to limited damage and quiescent infections .

Different types of quiescence have been described: delay of conidia germination or growth arrest after germination , square plastic plant pot endophytic symptomless growth in the apoplast , colonization of abscising flower organs followed by growth into ovaries or receptacles where growth arrests . Independent of the type of infection, the pathogen generally enters a short asymptomatic, biotrophic phase at the beginning of the disease cycle . An aggressive necrotrophic phase commonly succeeds the quiescent or asymptomatic phase once plant organs start to senesce or ripen, during which B. cinerea causes rapid decay of the infected tissues . B. cinerea’s infection mechanisms have been studied in model organisms and further characterized thanks to the availability of high-quality reference genome sequences . The fungus is known to actively promote plant susceptibility by employing a variety of virulence factors . In early stages, B. cinerea deploys sRNAs and effector proteins to suppress premature host cell death and immune responses, which enables the fungus to establish inside the host and accumulate biomass prior to the necrotrophic phase . It was demonstrated that B. cinerea Dicer-like proteins DCL1 and DCL2 produce sRNAs that are secreted from fungal hyphae and translocated to the plant cell where they interfere with the host RNAi mechanisms to silence host immune response genes in Arabidopsis and tomato leaves . Some secreted virulence factors can lead to host cell death, like effector proteins, toxins and enzymes involved in reactive oxygen species production . B. cinerea can also secrete oxalic acid that lowers the pH of the host tissuesand stimulates the production and activity of fungal enzymes like pectinases, laccases and proteases . Furthermore, oxalic acid accumulation leads to Ca2+ chelation, which in turn weakens the pectin structures of plant cell walls and inhibits the deposition of callose . Other virulence factors are cell wall degrading enzymes that enable B. cinerea to cause plant cell lysis and loosen walls to facilitate tissue penetration .

The fungus is known to produce plant hormones or hormone analogues that may disturb the host’s cellular metabolism and immune responses. The relevance of these mechanisms for the capacity of B. cinerea to infect strawberry remains unknown.Grey mould in strawberries can result from B. cinerea infections of open flowers or by penetration of fruit receptacle tissues . In primary infections, B. cinerea infects flower organs during or right after flowering, allowing hyphae to grow into the receptacle . The sources of primary inoculum range from overwintering sclerotia to conidia or mycelium from infected neighbouring plants . Infected senescent petals, stamens and calyxes can facilitate primary infections in fruit . Histological studies have shown that even though styles are frequently infected, fungal growth appears to be strongly inhibited and never reaches the receptacle. In contrast, fungal growth in colonized stamens can reach the receptacle in some cultivars . Following infection of the unripe receptacle by B. cinerea, fungal growth is usually arrested and a symptomless quiescent phase occurs. The mechanisms that lead to quiescent infections are not yet fully understood. Proanthocyanins appear to induce B. cinerea quiescence in unripe fruit by restricting the activity of fungal enzymes like polygalacturonases that are necessary for aggressive infection of hosts . Even though PA content in fruit remains constant during ripening, increasing polymerization of PAs leads to lower inhibitory activity in ripe fruit . Similarly, anthocyanins might delay B. cinerea infections or cause quiescence . For instance, strawberries illuminated with white fluorescent light showed increased anthocyanin content and delayed development of grey mould . Reduced fruit decay has also been observed in raspberries with high pigmentation and in transgenic tomatoes that accumulate anthocyanins .

Other small phenolics, especially catechins, may have a role in quiescence. High levels of catechins inhibit fungal growth, and a decrease in catechins is correlated with a reduction of other anti-fungal compounds such as lipoxygenases . Interestingly, young and ripe fruit have low catechin concentration, suggesting that initial infections of young receptacles are possible because they do not yet accumulate enough catechins to stop colonization . B. cinerea quiescence is complex and involves additional factors besides the accumulation of phenolic compounds. It has been proposed that quiescence in unripe fruit is initiated by: lack of nutrients such as sugars from the host, presence of preformed anti-fungal compounds, unsuitable environment for fungal virulence factors . In unripe strawberries, factors from all three categories are present, including lack of available sugars , preformed anti-fungal compounds , and high activity of PG-inhibiting proteins . Induction of the necrotrophic phase in ripe strawberries could be triggered by changes in biochemical composition of the host tissues associated with the ripening process, such as increased sugar content, volatile production and alteration of plant defences . These modifications promote not only fungal growth but also host susceptibility, e.g. via the release of oxalic acid and efflux of toxins . During secondary infections, the fungus initiates the necrotrophic phase without quiescence . The sources of conidia for secondary infections can also be diverse, from senescent leaves to infected fruit . Conidia from B. cinerea-infected flower parts are major sources of secondary inoculum . It has been estimated that more than 64% of the strawberry infections result from organic fragments that are in contact with the fruit, such as petals and stamens . Contrary to other fruit , senescent flower parts often adhere to strawberries long enough to retain water films for at least 8 h, which is the time needed for B. cinerea conidia germination . Secondary infections can also result from nesting, which corresponds to direct penetration of mycelia growing on neighbouring plant organs such as infected leaves and fruit . Generally, secondary infections proceed rapidly and B. cinerea can complete its germination and infection as fast as 16 h post-inoculation with a rapid increase in fungal biomass at 48 hpi . Early responses of strawberries to infection include higher expression of the defence genes FaPGIP and FaChi 2-1 , 25 liter square pot whereas lower expression of the reference gene DNA Binding Protein – FaDBP indicates extensive cell death induced by B. cinerea at late stages of infection .Fruit ripening influences the susceptibility of strawberry fruit to B. cinerea . Strawberries are mostly resistant to infection in their unripe stage, where they restrict fungal growth by causing quiescence. However, in the ripe stage, strawberries are highly susceptible and decay rapidly. Fruit susceptibility to fungal disease increases as ripening progresses; hence, B. cinerea appears to promote susceptibility in unripe fruit by activating specific ripening-related processes . In tomato fruit, master transcriptional regulators of ripening have been shown to have different roles in disease susceptibility. For example, the activity of the tomato transcription factor NON-RIPENING favours B. cinerea infection . Strawberries are non-climacteric fruit with a ripening programme different from that of climacteric tomatoes. Thus, a deeper understanding of strawberry ripening regulation and how B. cinerea may modulate particular ripening events are pivotal to characterize the dynamics of the strawberry-B. cinerea pathosystem.

Recent transcriptomic studies of developing strawberries point out that ripening events start between the ‘large green’ and ‘white’ stages, and involve changes in cell wall composition, sugar metabolism, hormone biosynthesis and responses, pigmentation and antioxidant levels . Moreover, a general decrease of oxidative phosphorylation processes has been observed during strawberry ripening . Normal strawberry ripening involves a variety of biochemical and physiological processes, some of which are discussed below in the context of B. cinerea interactions.Ripening is associated with the disassembly of the fruit cell walls, which leads to tissue softening. Cell wall degradation benefits B. cinerea as it reduces mechanical barriers to infection and spread, increases the possibilities of bruising and provides the fungus with access to simple sugars as a carbon source . In strawberry, cell wall solubilization occurs early in fruit development when the walls start to swell . Cell wall solubilization correlates with an increase in fruit sugar content, resulting from polysaccharide breakdown. A decrease of acid-soluble pectins and the alcohol-insoluble fraction of cell walls occur during ripening, whereas the water-soluble content increases . The degree of pectin solubilization and depolymerization is highlyrelated to strawberry fruit firmness . Silencing of an endogenous pectin lyase gene in strawberry resulted in fruit with higher external and internal firmness, mostly due to low pectin solubilization, stiffer cell walls, and increased cell to cell adhesion . Besides PL, other enzymes that may have affected strawberry firmness include PGs, β-galactosidases, endoglucanases, α-arabinofuranosidases and β-xylosidases . In addition to the fruit endogenous cell wall disassembly, B. cinerea secretes an extensive array of CWDEs that target most polysaccharides in the fruit cell walls, particularly pectins . These CWDEs include fungal PGs, such as Bcpg2, a gene that is mainly active in the early penetration stage . The expression of B. cinerea PGs is dependent on the host species, the plant tissue, temperature and the stage of infection .During ripening, the content of sugar in strawberries increases and therefore can serve as nutrients for B. cinerea. In unripe strawberries, the main sugars are glucose and fructose with low concentrations of sucrose. Sucrose levels increase rapidly during de-greening and red colouring . In tomato, it has been shown that the Cnr mutant, which does not accumulate high levels of sugars is still highly susceptible to B. cinerea infection . This observation suggests that even though sugars may serve as a susceptibility factor, high sugar concentrations are not essential for B. cinerea infection. However, sugar content could still influence susceptibility to B. cinerea as specific sugars may serve as ripening initiation signals. For instance, sucrose regulates abscisic acid levels in strawberries, which are necessary for normal ripening and could influence fruit susceptibility as described below . Like other ripening-related events, B. cinerea can alter neutral sugar and sugar acid levels in the infected host tissues, mainly by degradation and depolymerization of cell walls. This was reported for infections in tobacco and Arabidopsis leaves, where the fungus degrades pectins to release the monosaccharide galacturonic acid . ABA biosynthesis during fruit ripening is triggered by a decrease in pH, turgor changes, sugar accumulation, and the switch of sugars from mainly glucose and fructose to sucrose . Effects of ABA on strawberry susceptibility to fungal disease have not been extensively studied, but down regulation of the ABA biosynthetic gene β-glucosidase FaBG3 has been reported to result in fruit with limited ripening and higher B. cinerea resistance . In tomato, ABA accumulation is related to higher pathogen susceptibility, probably via activation of senescence . During strawberry ripening, the increase of ABA is correlated with a decrease of auxin, which induces early fruit growth and expansion but is known to inhibit ripening processes . The role of auxin in fruit susceptibility seems to depend on the plant species, as indole acetic acid treatment in Arabidopsis leads to susceptibility, whereas IAA-treated tomato leaves and eggplant fruit show lower infection severity . Ethylene has a secondary organ-specific role in strawberry ripening, particularly in achenes and green and white receptacles .

These values are higher than for the majority of domesticated fruits commonly reported, closest to very sweet fruits like pineapple and papaya ,access date 19 April 2010. The mean value for °Brix increased 22% from wild to cultivated fruits, from 13.6 to 16.6. It is interesting to note that even the wild fruits are very sweet, which may have predisposed this species to early selection as a cultivar. The reduction we saw in levels of phenolics in the pulp is consistent with a human preference for fruits with less bitterness . However, while a reduction in phenolics may indicate direct selection for palatability, it may also reflect an indirect effect of selection on the production of larger, sweeter fruits. Rosenthal and Dirzo suggest that selection for yield may result indirectly in the reallocation of energy away from defense, because of physiological constraints and tradeoffs. While all these differences were significant, the Discriminant Analysis showed that the great majority of the variance distinguishing wild from cultivated classes was driven by fruit size, with sugar concentration also contributing. This suggests that humans may have selected primarily for increased fruit size in this species, a hypothesis that could be tested with ethnobotanical studies. ENVIRONMENTAL VS. GENETIC CONTRIBUTIONS Many of the traits we measured, black plastic plant pots such as fruit size or sugar content, could be influenced by environmental conditions. Cultivated trees are likely to experience less competition for light, water, and nutrients than do wild trees.

However, several lines of evidence suggest that there is a large genetic component to the traits that distinguish wild from cultivated trees. First, both classes of trees, but especially the wild individuals, came from a wide range of environmental conditions. Some of the wild trees are now in open areas because the forest was cut around them, or are on the edges of forest fragments and therefore have access to more light and possibly nutrients than wild trees in the center of the forest. When we categorize the wild trees for their location, we see no trend toward differences in °Brix between trees on edges or in the open vs. trees in closed-canopy forests . We do see evidence suggesting that edge fruits areslightly bigger, a difference of about 10 g; this is substantially lower than the 73 g difference between the wild and cultivated means. Second, despite year-to-year variation in environmental conditions such as timing and intensity of the dry season, we saw very high correlations between years in trait values, especially for traits related to fruit size. We have recently planted a broad sample of wild and cultivated genotypes in two field plantations in Panama, which in 10 to 20 years should provide a better estimate of the genetic contribution to variation in this species.For most traits, we found no difference in trait variance between wild and cultivated samples. However, we did find significantly greater variance among cultivated trees than among wild trees for fruit mass and seed number . In contrast, there was lower variance among cultivated trees for the concentration of phenolics in pulp.

Through the process of domestication, we expect to see genetic variation strongly reduced for “domestication genes,” those traits that are critical for bringing the species into cultivation, while genetic variation should increase for “crop diversification genes,” reflecting a range of preferences during selection . Domesticated plants often show more intraspecific variation than their wild relatives for traits of interest to people , even in cases where there is an overall reduction in genetic diversity due to a domestication bottleneck . The reduced variance we found for phenolics may indicate consistent selection against bitterness, while the increased variance for fruit size could possibly reflect a range of preferences by the distinct cultural groups that utilize this species in central Panama, including Emberá and Wounaan communities, as well as people of Hispanic and Antillean descent. As mentioned earlier, caimito in this region may also include some genotypes transported from the Antilles. Ethnobotanical studies aimed at understanding cultural preferences, selection targets, and selection intensity could help elucidate this issue. However, it seems more likely that the broad range in fruit size is generated by a lack of fixation of alleles associated with domestication due to a contemporary or historical influx of wild-type genes, as well as recombination among cultivated genotypes. Zohary and Spiegel-Roy have suggested that most fully domesticated tree crops are asexually propagated, at least in the Old World. This is thought to increase uniformity and reduce the production of unwanted intermediate forms, particularly as many tree crops are highly heterozygous and show an outcrossing sexual system. Chrysophyllum cainito is not propagated asexually in Panama.

The mating system of C. cainito has not been studied, but the species has small flowers that are pollinated by small bees , and we expect that it is primarily outcrossing like similar tropical lowland tree species . We suggest that the extremely wide range of variation in fruit size among cultivated phenotypes is what might be predicted for a fruit tree species in the early stages of domestication. This is consistent with the findings of Leakey et al. for the semidomesticated fruit trees Dacryodes edulis and Irvingia gabonensis. However, unlike those species, the distributions of traits in our cultivated samples of C. cainito did not show statistically significantly more skew than wild populations .We found a strong positive correlation between fruit size and both seed number and seed size. A positive correlation between fruit size and seed number has also been reported in sweet pepper and tomato , and for these species the genetic basis is known. The correlations that we observe in C. cainito may be due to pleiotropic effects, tight linkage of loci, or a combination of these factors. From a physiological perspective, fruit size may be directly related to seed number because the developing seeds are a source of auxin for the developing fruit; therefore, an increase in number of seeds results in a larger fruit . In addition, a general “gigas effect” in domesticates can produce an increase in organ size through increased cell number and/or cell size reviewed in Pickersgill 2007, which could also explain the positive correlation of fruit size and seed size/number observed in caimito. Therefore, even if human preferences were in the direction of smaller seeds, as is the case in tempesquistle , or fewer seeds, genetic and physiological factors may constrain seed size and number in cultivated C. cainito.Nearly one-third of the world’s food production is lost or wasted through the food supply chain, according to the Food and Agriculture Organization. Fungal pathogens are responsible for causing diseases like rots and molds that result in reduced product quality, shelflife, and market value, leading to significant losses of harvested fruits and vegetables in post harvest. Fungal pathogens can gain access to the fruit tissues in different ways: by establishing latent infections of flowers, exploiting wounds and natural openings , or by directly penetrating the host cuticle. Vector insects can also cause damage, which is a common entry point for pathogens. Additionally, mishandling and physical damage of the products during harvest, sorting, packing, transportation, cold storage, and retailing contribute to a higher incidence of disease. The germination of fungal spores requires moisture and stimulation from host solutes that diffuse into the initial penetration site. After germination, specific signals from the plant surface trigger the formation of specialized fungal structures that enable the pathogen to penetrate the plant cell walls and establish infection. Once the disease is well established in one fruit, black plastic planting pots it spreads quickly to adjacent healthy fruit, a process known as nesting. A well-known nesting pathogen is Botrytis cinerea, which releases airborne conidia that readily nest on damaged or senescent fruits, initiating decay and facilitating further spread. Rhizopus stolonifer, the causal agent of Rhizopus rot in various fruits and vegetables, is another prominent example of a nesting pathogen. Following spore germination, R. stolonifer produces mycelial stolons that attach to the host surface, enabling it to colonize healthy fruits and initiate infections. Penicillium spp. are commonly considered wound-dependent pathogens; however, it is commonly observed that if the fungi are initially established in a rich food source like a decaying fruit, the mycelium can readily invade the tissues of an adjacent healthy fruit. This phenomenon is how an initial low incidence of green or blue mold in a packinghouse storage facility can develop into major losses after prolonged fruit storage. Integrated pest management strategies have been developed to reduce or eliminate fruit infections, including synthetic fungicides before and after harvest, biological control agents, essential oils, cold storage, and modified atmosphere packaging.

To test the effectiveness of these strategies, reliable laboratory or field-based inoculation methods are required to obtain quantitative data that goes beyond subjective ordinal rating scales that may be influenced by human bias. While natural infections can provide insights into disease dynamics in real-world scenarios, they can be unpredictable and impacted by environmental factors, making it difficult to control and replicate experimental conditions. Therefore, the study of plant-pathogen interactions generally relies on pathogen inoculation techniques. Dip and spray are two common inoculation methods where fruits are covered in a fungal spore suspension by submersion or application with an atomizer. These methods allow for uniform, whole-fruit inoculation but may result in lower disease incidence and severity due to the challenges in standardizing the process. Another method is wound inoculation, which involves creating artificial entry points on the fruit surface before applying the fungal spore suspension or mycelial plug. This method simulates wounds and enables precise and reproducible experiments. Still, it may not accurately represent the natural infection process as it bypasses the initial steps of adhesion and penetration on the plant tissues. As of present, our comprehensive literature review has not revealed any reported methods that faithfully replicate the post harvest nesting phenomenon. This study presents a novel methodology for assessing post harvest infections of persistent fungal pathogens through contact-based inoculation of fruits. We optimized this protocol using four impactful fungal pathogens and post harvest commodities that are commonly affected by fungal disease: Botrytis cinerea and Penicillium expansum in tomato and apple, respectively, and Penicillium italicum and Penicillium digitatum in orange. B. cinerea is the causal agent of gray mold, a devastating disease that causes billion-dollar losses on fruit commodities worldwide. P. italicum and P. expansum, causal agents of blue mold, and P. digitatum, causal agent of green mold, are significant post harvest diseases. Our protocol unveils new possibilities for testing disease management strategies and studying the nesting behavior of post harvest fungal pathogens.We conducted several trials to determine the best source of fruit tissues for contact-inoculation. The developed inoculation method involves producing the source fruits through wound inoculation, and preparing the non-wounded target fruits. The selected source and target fruits ideally should not have any surface imperfections such as scars, wounds, or bruises, and apples should have an intact pedicel. Fruits were first disinfected in 10% sodium hypochlorite, rinsed twice in sterile Milli-Q, and dried with sterile tissue paper before inoculation. Source fruits were wounded multiple times on the stem end. For oranges, three equidistant wounds were created using a sterile nail following the protocol developed by Vilanova et al.. For tomatoes and apples, equidistant wounds were created in four locations with a sterile pipette tip . Oranges were inoculated with P. italicum or P. digitatum, while tomatoes and apples were inoculated with B. cinerea and P. expansum, respectively. Source fruits were incubated under high relative humidity at 10 °C for 10 and 13 days for orange, 20 °C for 4 days for tomato, and 10 days for apple. By then, the source fruits should have developed lesion sizes of about 30, 10, and 20 mm for oranges, tomatoes, and apples, respectively. In initial attempts, we used culture media plugs with well-established fungal mycelial growth as the inoculum source. However, this method was not always successful and did not accurately mimic how fungal infections occur while handling and storing fresh produce.Two different inoculum sources were explored for the contact inoculation: whole fruits and fruit tissue sections. When using whole fruits as the source, target fruits were placed on a plastic boat lying on the equatorial region.

The presence of nuclear fluorescent patterns however, clearly shows that at least some hybrid fluorescent proteins are actively transported into the nucleus Surprisingly, the addition of an extra NLS tag in the pWUS:NLS-eGFP-WUS reporter did not significantly enrich the nuclear localized pattern compared to pWUS:eGFP-WUS. Such a pattern might be expected to occur if WUS has a native NLS motif, which would make the added NLS tag is functionally redundant. The present data however, cannot rule out the possibility that NLS tag is blocked by some aspect of WUS structure. The NLS-eGFP-WUS protein is 8% larger than eGFP-WUS, and this figure is reminiscent of the 15% reduction found in pWUS:NLS-eGFP-WUS meristems, and might suggest that the limited mobility of the NLS construct reflects a size-dependent fractionation process, rather than nuclear trapping. The continued presence of WUS in the cytoplasm however, requires the existence of a nuclear export mechanism to balance out the effects of nuclear import. Although such a function has not been attributed to the WUS protein, the EAR-like motif present in its C-terminus closely resembles a lysine rich NES motif. The same motif is also recognized by TPL, though it is not clear which function predominates in WUS. Even if EAR-like motif functions as a nuclear exit sequence however, draiange planter pot a system based exclusively on nuclear pore transport would likely shift the equilibrium to one extreme state or another rather than be perfectly balanced at an intermediate state.

One clue about how a stable intermediate is achieved comes from the pWUS:eGFP-NES-WUS construct, whose fluorescent pattern is very weak, but is not zero , suggesting that WUS is rapidly degraded in the cytoplasm. This observation also indicates that WUS EAR-like motif has at best a weak NES function, as the added NES tag could only have such a strong impact if the native WUS molecule lacked a strong native NES function.Together these observations indicate that only small fraction of WUS proteins are transported into the nucleus, while those that remain in the cytoplasm are degraded. Rather than being nuclear enriched, this situation is probably more accurately described as cytoplasmic depletion. Interestingly, the WUS subcellular pattern closely parallels a similar situation for Cytidylyltransferase proteins in the Mouse model, where mono-ubiquitination of the NLS motif was demonstrated to prevent nuclear import, and was further associated with higher rates of proteolytic degradation of CCTα in the cytoplasm.On the basis of current evidence, it seems likely that cytokinin responses are necessary for WUS protein stability. This may reflect a common trend for SAM expressed genes, as cytokinin exposure is also known to increase the stability of ACS and ARR1. Evidence of a positive relationship is perhaps best seen in the ahk2/3/4 receptor mutant background, where WUS mRNA is transcribed normally in the complete absence of cytokinin responses , yet the translated GFP reporter is barely detectable . Conversely, when cytokinin responses are induced by pCLV3:GR-LhG4 x ARR1ΔDDK-GR, we see an increase in WUS proteins in the L1 and L2 cells after 48 hours, but little or no WUS transcription in these cells . This suggests that the WUS proteins that move into the L1 and L2 cells are rapidly degraded in the absence of strong cytokinin responses, but become protected following ectopic cytokinin activation. However, the idea of a positive correlation begins to break down when p35S:GRLhG4::p6xOP:CKX3 induction is considered, because this did not obviously reduce pWUS:eGFP-WUS fluorescent levels .

The fact that the peak fluorescence shifted down by one layer might indicate that the response-free zone became larger, but the variability of this construct makes it difficult to draw firm conclusions. Another potential problem can be found in pCLV3:GR-LhG4 x ARR1ΔDDK-GR x pWUS:eGFP-WUS plants that have been induced with dexamethasone for 48 hours. Although cytokinin responses are homogenously distributed in these meristems, the pWUS:eGFP-WUS pattern does not clearly show strong WUS expression in the peripheral regions where cytokinin induced stability might be expected. The results of cycloheximide and MG132 treatments do not help clarify this situation, as the alternating patterns of stability and instability cannot easily be explained in terms of the cytokinin signaling pathway alone. To do so requires assuming that the cytokinin phosphor-relay system has a previously undetected branch pathway, potentially regulating a protease with equally unusual phospho-dependent activity. However, this model is not much different than the observation that cytokinin influences WUS stability through both protein translation protein pathways, as both models require multiple steps with poorly known intermediates. Attempts to identify the possible intermediates using lists of cytokinin-targeted genes do not clearly help resolve this situation, as a meta-analysis found only five translation related genes, two of which modify mRNA, one that modified tRNA, and two that are involved with ribosomal RNA processing. The same list of cytokinin targets also contains six protease genes , while a single representative from the ubiquitin/proteosome pathway was down-regulated. In the absence of a clearly direct cytokinin-WUS connection, it is quite tempting to speculate that protein stability is a secondary effect of cytokinin responses. If so, stability may be a generic feature of cytokinin responses, which has the potential to affect all proteins simultaneousl.Experiments with auxin on the other hand, suggest a much more direct link with WUS stability. Four hours of exogenous NAA treatment dramatically reduced pWUS:eGFP-WUS fluorescent levels, while comparable treatments with cytokinin took a minimum of 12 hours to show the slightest response in WUS expression.

The auxin–induced degradation was also readily blocked by cycloheximide treatments , indicating that the response requires protein translation. Still, exactly which proteins are translated, and how they affect WUS stability is not clear. Auxin induced degradation may have a functional significance for lateral anlagen though, as the concentration of auxin responses in distinct foci, would help rapidly reprogram the anlagen cells by degrading conflicting developmental proteins. This hypothesis is consistent with the large marginal voids of WUS and CLV3 expression found when cytokinin responses are ectopically induced with the pCLV3:GR-LhG4 x p6xOP:ARR1ΔDDK-GR system , which were often correlated to the presence of leaf primordia and sites of auxin accumulation. Similar marginal voids can also be seen in WT meristems treated with exogenous cytokinin. Although not quite as direct, other research has also shown that WUS transcript levels are indirectly linked to auxin transport. In addition callus tissue studies have found that induction of SAMs does not require cytokinin alone, but instead requires an appropriately high concentration of auxin or a balanced auxin/cytokinin ratio, clearly implying that auxin is a significant part of the process. Considering the overall organization of the SAM, this suggests a model where WUS helps stabilize the mutually exclusive pattern of auxin and cytokinin responses in the PZ and RM by activating the biosynthesis of both hormones and auxin transport genes within the CZ. The lack of hormone responses in the very cells that produce them is consistent with a similar pattern in root development , and given the often symplast-like environment in the SAM, a repressive mechanism may be necessary to prevent hormone response proteins from spreading into the CZ and suppressing biosynthesis. The fields of protein stability and instability brought about by the hormone responses also appears to define the number of WUS producing cells, and eventually, plant pot with drainage the concentration of WUS molecules that reach the CZ, forming an indirect, but stable set of feedback loops that share WUS as an anchor. The CLV3 pathway may represent another feedback loop within this framework, as it is also activated by WUS in the CZ, similar to the postulated activation of hormone biosynthesis genes. Although the intermediate steps are not clear, CLV3 appears to suppress cytokinin-induced proliferation, as seen by the hypersensitive response of clv3-2 mutant to exogenous cytokinin . By doing so, it may potentially function as a third feedback loop, negatively regulating WUS transcription though a mechanism that is slightly more direct than either hormone pathway alone. It would thus be of great interest to learn what proteins regulate WUS transcription in the RM, as the ahk2/3/4 RNA in-situ clearly shows that cytokinin responses are not involved. Intraspecific variation in seed dispersal has important consequences for individual reproductive success, plant population dynamics, community structure and evolution. For example, intraspecific variation in seed dispersal distances , the microhabitat destination of dispersed seeds and the treatment in the mouth and gut affect demography and individual plant fitness through their impacts on the number of seeds dispersed, surviving, germinating and growing as seedlings.

As a prominent example, dispersal kernels that include inter individual variation in dispersal distances are not equal to a population-level dispersal kernel based on mean dispersal distances. Including this intraspecific variation can alter the rate of population spread and the extent of gene flow . Furthermore, individual variation in seed dispersal increases the range of habitats and conditions where seeds are dispersed, increasing the likelihood of the population to persist under unfavourable events . Although poorly studied, intraspecific variation in seed dispersal may also influence community assembly, species richness and responses to anthropogenic changes . See Snell et al. for a thorough review of the consequences of intraspecific variation in dispersal. However, given the historical focus in seed dispersal studies on population means, there are large gaps in our understanding of intraspecific variation in dispersal. We do not know how pervasive detectable variation in seed dispersal is, what the drivers of individual variation are and to what extent drivers have independent versus interactive effects. To date there only have been scattered efforts to summarize the breadth of our understanding of the drivers of intraspecific variation in seed dispersal. The phrase ‘intraspecific variation in the drivers of seed dispersal’ is diffuse and subsumes multiple types of drivers and levels of variation. Decomposing this variation helps structure our thinking about intraspecific variation in dispersal. First, drivers of intraspecific variation in seed dispersal can be categorized as intrinsic variation based on trait expression of individual plants and extrinsic variation based on the ecological context of the plant . Further, intraspecific variation can be divided into variation among individuals and variation within individuals . Most drivers of intraspecific variation in seed dispersal have both an inter individual and an intra individual component . When considering drivers of intraspecific variation in seed dispersal, it is important to clarify what aspects and consequences of dispersal are being affected. Seed dispersal effectiveness, or SDE, depends on both the quantity of seeds dispersed and the quality of dispersal provided to those seeds . While SDE is usually viewed as mean quantity multiplied by mean quality, these means are derived from a sample of individuals that likely differ substantially in both the quantity and the quality of dispersal. Beyond SDE, the probability of long-distance dispersal can vary intraspecifically, which in turn contributes to population spread and gene flow. In this review, we focus mostly on seed movement, largely because that is what we have the most information on. However, we address consequences for seedling establishment or recruitment where relevant information is available. In this paper, we provide a broad but not exhaustive review of the drivers of intraspecific variation in the quantity, and to a lesser extent, the quality components of seed dispersal . We emphasize intrinsic drivers and inter individual variation because of our interest in individual fitness, defined as the contribution of an individual to future generations . However, we also consider intra individual variation in traits because it can scale up to affect inter individual variation in dispersal. Further, intra individual variation is not independent of inter individual variation. Lastly, we consider simple intraspecific variation in traits because much relevant work focuses on population-level trait variation without considering its apportionment into intra and inter individual components. We have several goals with this review. First, we illustrate the breadth of drivers of inter individual variation in seed dispersal. Second, we use diverse examples to illustrate the broad geographic and taxonomic scope of inter individual differences in seed dispersal, to assess how consistently they occur and to explore the range of impacts on seed dispersal processes. Third, we briefly discuss the barriers to fully understanding these drivers and their effects.Crop size varies substantially among individuals and populations within a season and across years . Crop size is probably the most widely studied and best-supported driver of inter individual variation in the quantity of seeds dispersed.

This division rate, along with the appearance of new organs on the periphery, have long served as landmarks to identify the meristem structure, and they are conveniently known as the Central Zone and PeripheralZone . Typically the CZ is further subdivided into upper and lower portions, such that cells in the L1 and L2 are recognized as the “upper” CZ, while those in the underlying L3 are known as the Rib Meristem . The astute student however, will note that this interpretation is not quite universal, as some researchers further postulate the existence of a fourth “Organizing Center” inserted between the CZ and RM tissues. Although not shown in Figure 2.0, the OC is equivalent to the rounded apex of L3, pushing the remaining part of the RM somewhat deeper into the stem. Until better genetic evidence is available though, only CZ, PZ, and RM will be used for the remainder of this dissertation. While the numbering system shown in Figure 12 does provide a useful set of spatial coordinates, it is also somewhat misleading as it implies that the SAM is static structure, unchanging over time. This could not be further from the truth. Instead, it must be remembered that the SAM is a site of plant growth, and as a result its cells are in a state of constant flux as they divide, grow, and differentiate. For example, the repeated perpendicular divisions that occur in L1 and L2 actually cause these layers to expand sideways, drainage pot where the displaced cells eventually bend around the curve of the apical dome and become part of the cylindrical stem surface.

The motion is reminiscent of the path taken by water droplets in an umbrella-shaped fountain, though the individual plant cells move considerably slower. If growth by lateral displacement is followed to its logical extremes, it is important to note that all of the founding cells will be pushed off to the sides over time, while new ones take their place in the middle. No single cell in the SAM is a permanent resident. The overall shape and size of an SAM is perhaps more analogous to a standing wave, where stability is the illusion caused by a dynamic equilibrium. Maintaining that wave is of course a difficult challenge, as the inputs to that equilibrium must be precisely matched to its outputs at all times. Failure todo so would quickly rob the plant of its ability to grow, with obvious consequences for survival. Exactly how this balance is maintained is not fully understood, but the motion of the cells makes at least one part of the process perfectly clear: the cells must change their identity as they are moved from one place to another. Those that start in the CZ for example, switch to PZ gene expression patterns as they move further away from the middle, and may later adopt leaf and flower identities as they are incorporated into mature organs. The ability of a cell to determine its location within the SAM structure is thus of paramount importance, yet it must do so in the absence of any stationary reference point. So far as currently understood, each cell solves this problem in exactly the same way a person would do so: it talks to its neighbors. Based on what the individual cell sees and what its neighbors report seeing, it is possible to work out exactly where the cell is located in the overall plant structure. Of course in actual plant tissues such communication occurs largely through to the exchange of proteins, hormones, and RNA molecules, though increasingly evidence suggests that mechanical forces in the cell wall may also contribute some information.

Some molecules can travel further distances than others, some are modified en-route in order to become functional, and still others move from cell to cell in precise patterns, much like the knight in a game of chess. When these molecules are produced in different areas of the plant, the surrounding cells can estimate their relative locations to each other simply by reading the chemical bar-code in their local milieu, and then develop accordingly.At the present time, only a few such routes of chemical communication have been identified, two of which are plant hormones: auxin and cytokinin. Auxin is best known for increasing the volume of cells, though it also has roles in apical dominance and tropism growth patterns. Cytokinin meanwhile is known for stimulating cell division, in addition to other roles in senescence and pathogen responses. Together the function of the two hormones would appear to complement each other very well in terms of overall growth, yet within the SAM they appear to mix about as well as oil and water. Cells that respond to auxin often don’t respond to cytokinin, and vice versa. Why this should be so is not well understood, but studies of root vasculature development suggest that their mutual exclusion is actually used to generate spontaneous patterns that help guide plant development. In callus tissue, the two hormones are often found to have response patterns arranged in a polka-dot like arrays, where each hormone “dot” is surrounded by a circular field belonging to the other. The SAM is organized around a single such dot, where cytokinin responses occur in the RM, and auxin responses occur in the PZ which often occur in discrete foci corresponding to new lateral organs. The CZ cells in contrast, do not appear to be sensitive to either hormone, but instead express both auxin and cytokinin biosynthesis genes.

The production of cytokinin in the L1 and L2 is also consistent with the distribution of bioactive cytokinin concentrations observed with immunological techniques and with GFP reporter systems . This suggests a stable arrangement of three mutual exclusion zones within the SAM, which closely correspond to the known CZ, PZ, and RM tissues. Root apical meristems in contrast, appear to be based on the reciprocal arrangement, as roots have an auxin response dot in the middle surrounded by cytokinin responses in the overarching root cap, concentrated in the root cap columella cells.There is also evidence of a more complex set of feedback loops, as WUS has been found to regulate components of the cytokinin signal transduction pathway, and exogenous cytokinin are able to stimulate WUS transcription. Altered cytokinin signalling pathways have also been shown to affect CLV3 expression patterns . WUSCHEL-LIKE HOMEOBOX5 , which is closely related to WUS, is known to participate in auxin pathways within the root, while the generation of SAMs from callus or root tissue has repeatedly been shown to require a pre-incubation on auxin rich media, where it may actually stimulate auxin transport. Micro RNA molecules may also be involved, as a variant of AUXIN RESPONSE FACTOR 10 that was resistant to miR160a was able to increase WUS and CLV3 expression patterns. Clearly, there is a lot going on. To help clarify how such cross-talk contributes to SAM structure, large pot with drainage the research presented in this dissertation explores two closely related subjects. The first is the regulation of CLV3, which was studied by resolving the promoter structure of this gene in chapter 3. The results suggest that CLV3 is regulated in part by auxin responses, while activation and/or repression is likely to be controlled complicated set of cis-motifs in the 3’ enhancer region. The presence of these 3’ motifs in a known transposon also suggests a novel origin of the WUS/CLV3 feedback loop. Chapter 4 meanwhile, explores the possibility that WUS and cytokinin responses form a second feedback loop necessary for SAM structure. This was done by narrowing down the possible cellular and biochemical routes by which cytokinin could affect WUS transcription, translation, and protein movement. The results however, suggest that the two pathways are atlargely independent of each other, though cytokinin responses may increase WUS stability in the RM. Unexpectedly, the data also found that the absence of cytokinin responses in the CZ is a critical part of SAM structure. The cytokinin response-free cells were also found to have an enhanced protein degradation mechanism, which may help shape the WUS protein gradient. Interestingly, WUS proteins were found to be rapidly degraded following auxin treatments, suggesting a model in which the SAM structure is defined by cytokinin-induced stability in the RM, and auxin-induced protein degradation in the surrounding CZ and PZ cells.The WUS-CLV3 feedback loop has long been an attractive model to explain how SAM structure is maintained in a dynamically changing cellular environment. Simply by combining activation of CLV3 with the repression of WUS, computer simulations have repeatedly shown that this is sufficient to maintain constant population of cells with CZ and RM identity.

However, despite the simplicity of this model, the molecular mechanisms that carry out the feedback loop have instead revealed a number of potential complications. On the forward path for example, WUS is known to be a bi-functional transcription factor, activating and repressing several hundred different target genes. Currently it is not currently known exactly how WUS switches from activator to repressor, but it has been shown to directly bind to DNA motifs in AGAMOUS and CLV3 regulatory regions, where it activates their transcription. Additional binding sites on repressed targets such as KANADI1, YABBY3, ASYMMETRIC LEAVES2 have also been identified. Complicating this model of is the observation that CLV3 activation requires both WUS and SHOOTMERISTEMLESS in leaf tissues, suggesting that the presence of WUS alone is not sufficient. In addition WUS has also found to directly interact with the GRAS domain transcription factor HAM1 , as well as the potent transcriptional repressor TOPLESS. TPL itself further has been shown to assemble a protein complex with Sin3 ASSOCIATED PROTEIN and HISTONE DEACETYLASE 19, suggesting a potential link between WUS and chromatin modification. In order to discriminate between the two models, this study began by attempting to identify the cis-regulatory environment around the CLV3 locus. The CLV3 expression pattern was first carefully recorded with a GFP reporter, which in contrast to previously published RNA in-situ’s, found layer-specific differences in CLV3 transcriptional output. The regulatory regions of CLV3 were then annotated by mapping predicted transcription factor binding sites and computationally significant cis-motifs, which were further resolved with phylogenetic foot printing. This analysis found that CLV3 has a very simple 5’ promoter, containing an auxin responsive element, suggestive of ubiquitous expression. The 3’ enhancer in contrast, contained at least 3 large cis-regulatory modules, two of which were found within a naturally occurring transposon, while the 3rd included several known WUS binding sites. On the basis of promoter deletion experiments, all three cis-regulatory modules were found to be required for CLV3 activation. The existence of the transposon in turn, has several implications for the evolution of the WUS-CLV3 feedback loop and Brassicaceae plant anatomy.Previous reports of the CLV3 expression pattern have consistently found it localized to the apex of the SAM, where it is often used as an indicator of CZ cell identity. Within this region, the expression pattern is somewhat variable, as previous RNA in-situ revealed a narrow inverted cone-shape, while GFP and GUS reporters often produce more indistinct rounded shape 3-4 cell layers deep. In contrast, the present study found a slightly more complex pattern when viewed as a longitudinal section. In perfectly centered sections, the pCLV3:mGFP5-ER reporter often appears in an inverted cone shape, but the expression zone is noticeably broader than the previous RNA in-situ results . As the section plane is displaced from the central axis and becomes more tangential, a conspicuous gap is frequently visible, where the L2 cells have less fluorescence than those immediately above and below. This suggests a bi-partite expression pattern where a flat, circular domain occurs specifically in the L1, and a second spherical domain occurs underneath in the L3 cells . In order to identify the CLV3 regulatory structure, this work began by annotating all known regulatory motifs on an 8kb genomic sequence centered on At2g27250. Transcription factor binding sites were identified by consulting multiple online prediction tools which quickly found over two hundred predicted cis-motifs, many of which had low probability scores. The odds of identifying functional cis-motifs were increased in a few select cases by adding 5bp sequences on either side of the core motif, based on previously identified target sites for WUS, ARF1, and ARR1.

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 . Fungal plant pathogens are also known to express inhibitors of these types of proteases as a counterdefense . 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, greenhouse vertical farming 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, 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. However, we also observed upregulation of biosynthetic genes involved in production of trichothecenes , nft vertical farming which indicates that F. acuminatum also relies on other toxins during infection of tomato fruit concordant with the classification of F. acuminatum as strong toxin producer . Additionally, the AA6 family that appears during RR infections of F. acuminatum and R. stolonifer may be involved in metabolism of host defense compounds. These enzymes are 1,4-benzoquinone reductases, which have been shown to function in fungal protection against destructive host-produced quinones . Another physiological factor which may influence the success of infection is the pH of the pathogen-host interface. As the tomato fruit ripens, the apoplast becomes more acidic . Furthermore, B. cinerea has been shown to acidify the host environment through the production and secretion of oxalic acid . A key enzyme in oxalic acid biosynthesis is BcOAH1 , which encodes oxaloacetate hydrolase . However, there is significant down regulation of this gene in RR fruit compared to MG fruit. This suggests that, if B. cinerea utilizes oxalic acid to acidify tomato fruit, it does so to a much lesser extent in RR fruit where the pH is already comparatively acidic. In contrast, during infection of Arabidopsis roots, F. oxysporum relies on alkalinization via peptides known as rapid alkalinizing factors . However, a BLAST search of RALF sequences, as was performed to identify fungal RALFs in Thynne et al. , revealed no clear RALF genes in our transcriptome of F. acuminatum.

The importance of fruit ripening for the success of fungal infections was confirmed by comparing fungal growth and disease development in fruit from wild-type and a non-ripening mutant after fungal inoculation. Growth and morphology of B. cinerea, F. acuminatum and R. stolonifer on nor MG and RR-like tomato fruit was comparable to that on wild-type MG fruit. This result is in agreement with our previous report that nor tomato fruit is resistant to B. cinerea infections . The inability to infect non-ripening tomato fruit highlights the dependency of these fungi on the activation and progression of ripening events that transform the host tissues into a favorable environment for disease development. Altogether, our results confirm that infection success of the three pathogens B. cinerea, F. acuminatum and R. stolonifer largely depends on fruit ripening stage. This is due to all three pathogens sharing similar lifestyles and necrotrophic infection strategies. However, the capacity to infect different plant tissues differs between the three fungi. B. cinerea shows distinct strategies in both ripening stages likely due to its ability to induce susceptibility in the host , whereas R. stolonifer is active almost exclusively in RR fruit. The ability of F. acuminatum to infect both MG and RR fruit may be reflective of its especially wide host range, which includes insects in addition to fruit . A summary of infection strategies utilized by the three pathogens during infection of MG and RR tomato fruit is shown in Table 2. Further research on which processes identified are required for successful infection would lead to a greater understanding of fruit-pathogen interactions and, ultimately, strategies for their management.In the simplest possible terms, Alternate Bearing is a two year cycle of fruit production that occurs in perennial plants, and is best known from fruit-bearing trees like apples and avocados. In one year, the plants will produce many fruits, while in the second year, they produce very few. This biennial pattern then often repeats itself in the following years creating a repetitive cycle, revealing itself as a saw-toothed line when fruit yields are plotted over many years . The fluctuation in yield is usually quite moderate and only rarely reaches the extreme values of 0% and 100%, but even minor variations can readily be detected in commercially grown orchard trees. The phenomenon is not limited to trees though, as similar biennial cycles have also been documented to occur in perennial herbs, monocots, and forest trees, which suggest that alternate bearing is a fairly common, if seldom seen plant behavior. From an economic standpoint though, the presence of alternate bearing in commercial orchards is considered to be undesirable for many reason. Not only do alternating trees tend to produce less fruit on average than regular-bearing varieties, the resulting fruit often display characteristic variation in size and appearance that reduces their market value. For example, large crops tend to produce small and poor quality fruits, while small crops can occasionally produce large and abnormally swollen fruit. Many fruits are sorted into sizes according to their intended market, where this variation makes it more difficult for the farmer to produce sufficient numbers of each. Harvest costs are largely the same whether the crop is large or small, and large crops often incur extra processing and storage fees when they can’t be sold immediately. In addition, alternate bearing trees are prone to synchronization, during which all of the trees in the orchard fluctuate in lock-step with each other. Frequently attributed to a late flower-destroying frost or other temperature anomalies, such synchronization can occur over a wide range of scales, ranging from isolated trees, to whole orchards, and even entire geographic areas . Fruit production in synchronized areas tends to saturate the market with low-quality fruit in one year, and provide little or no fruit in the second, thus limiting the potential profit in both years of the cycle. Although there are horticultural techniques available to force the trees to become regular bearing again, these introduce additional expenses in the form of time, labor, and materials. Thus the combined effects of yield, quality, irregular production, and additional expenses can easily make alternate bearing crops uneconomical to produce, despite any other attractive qualities they might have.

Our analysis of Solanaceae euFUL homologs show that FUL1 and FUL2 are broadly expressed in leaves, flowers, and fruit . This overall similarity in expression may indicate a conservation of cis-regulatory elements in gene copies following duplication . Supporting this, our investigation into the number of putative TF binding sites in the promoter region of euFULI homologs did not reveal statistically significant differences . In tomato fruit development, FUL1 expression increases with time, whereas FUL2 expression reaches a maximum at early stages and then decreases over later stages . This variation in expression associated with the developmental stages might be due to changes in cis-elements as a result of the accumulation of random mutations over time . Our analysis did find differences in the number and location of predicted binding sites for specific TFs or families, for instance for ARF, STK, and EIN3 TFs, which may account for the types of differences in expression seen between euFUL paralogs. The 5 kb region upstream of the FUL1 transcription start site in tomato contains three putative ARF binding sites but the corresponding region of FUL2 in tomato contains no such motifs . ARF TFs, raspberry cultivation pot important in tomato fruit development, are activated in response to auxin and may upregulate or repress downstream genes ; the absence of binding sites from the FUL2 promoter is the type of factor that might underlie differences in expression observed between FUL1 and FUL2. Predicted STK binding sites are only found in the promoters of potato FUL1, tomato FUL2 and woodland tobacco MBP10.

STK and STK like proteins appear to function in storage protein synthesis, glucose reception, and vegetative and reproductive development . Meanwhile, the 2 kb upstream region of FUL2 contains a putative site for EIN3. This protein is involved in the development of tomato in response to ripening-associated ethylene production . No such motifs are found in the corresponding region of FUL1. In contrast, the 2–5 kb region in FUL2 contains four putative sites for EIN3 while the corresponding region in FUL1 contains three such sites . Such variation in number and location of TF binding sites has been shown to be associated with the temporal differences in gene expression . Whereas the euFULI members largely overlap in spatial expression with some variation associated with developmental stages, the euFULII homologs show less consistent spatial expression patterns. Only MBP20 is expressed in tomato roots and potato fruit while only MBP10 is expressed in potato tubers . However, these “on” or “off ” expression patterns cannot be explained by the presence or absence of any putative TF binding sites . These two paralogs, which appear to be the result of a tandem duplication and inversion, are located approximately 14.3 Mbp apart on chromosome 2. Although gene clusters resulting from tandem duplications are often coexpressed, this is not the case when there are large physical distances between the genes . An investigation into the expression of human transgenes in mice also found changes in expression as a consequence of an inversion, possibly through disrupting enhancer activity or changes to chromatin structure . Chromosomal rearrangements such as inversions may also result in novel connections between coding regions and other promoters or long distance regulatory motifs while disrupting the original regulatory mechanisms . This sort of re-coupling of one of the two paralogs might lead to the types of contrasting expression patterns observed for MBP10 and MBP20.

However, the expression patterns are not consistent across species and this might be due to additional changes following the inversion . An in-depth analysis of the entire loci and their genomic environment for all paralogs in multiple species would be necessary to determine if the tandem duplication and inversion are associated with changes in proximity to heterochromatin, additional rearrangements, or other phenomena that might have altered gene expression.The first intron of some MADS-box genes contains cis-elements important for the regulation of expression . Studies have found that deletions in the first intron of a FUL-like gene in Aegilops tauschii alters expression and results in the loss of the vernalization requirement . Consistent with this, the first introns of angiosperm euFUL orthologs are generally in the range of 1–10 kb . In contrast, tomato MBP10 has a short first intron of 80 bp. We compared the putative TF binding sites in the first introns of MBP10 and MBP20 in tomato to characterize potential loss of such sites, which might suggest reduced gene regulation. The first intron of MBP10 is predicted to have no TF binding sites, while the first intron of MBP20 is predicted to contain 88 TF binding sites . These included binding sites for MYB, HSF, Dof, WRKY, and MADS-box TFs. Specific TFs predicted to bind to these sites include MYB2 and C1 , which play roles in anthocyanin accumulation and lignin biosynthesis, PBF , which plays a role in endosperm storage protein accumulation, and SPF1 , thought to function in fruit ripening . A similar pattern was found in analysis of the first intron of MBP10 in Nicotiana obtusifolia, which is 110 bp . This analysis found three putative TF binding sites for MYB2 and one for PBF. By contrast, the first intron of N. obtusifolia MBP20 is predicted to have 133 TF binding sites and include a repertoire similar to those found for tomato MBP20.

To determine whether the difference in TF binding site number between the paralogs represented a gain of sites in the MBP20 genes or a loss in the MBP10 genes, we also searched for TF binding sites in the first intron of AGL79, the single euFULII ortholog in A. thaliana . We found that it contains 49 predicted TF binding sites for five different TFs in four families: MYB , HSF , WRKY , and GT-box . Although this number is substantially smaller than the number of sites predicted in the first introns of the Solanaceae MBP20 genes, the results suggest that there has been a loss of TF binding sites in MBP10. Core-eudicot euFUL and basal-eudicot FUL-like genes frequently have broad expression patterns and are generally expressed in fruit . Therefore, the absence or extremely weak expression of MBP10 in fruits of all species, and its weak expression in most organs of tomato and potato is notable . This relatively weak expression may at least in part be due to the loss of TF binding sites in the first intron and suggests a potentially reduced role in regulating fruit-related developmental processes. Importantly, the loss of putative TF binding sites and low expression, combined with the faster evolutionary rate, suggest MBP10 might be in the process of becoming a pseudogene. Further support for this hypothesis comes from an examination of the MBP10 sequences, which suggests that at least two of the sequences in our study show a frame shift that would result in an premature stop codon.There are many ways to train and prune deciduous fruit trees, and no single method is right for all situations and needs. When selecting fruit trees, one important consideration is the desired size of the trees at maturity. Many people prefer small trees because they are easier to manage and harvest and because more trees can be grown in a limited space. Other people prefer full-sized trees because they provide more shade and more fruit per unit area.Genetic dwarf trees usually produce very short internodes , resulting in compact branches with dense foliage. These trees grow to about 8 to 10 feet tall and wide at maturity. They make beautiful landscape trees that are easily managed to provide adequate amounts of fruit for a single family. Excellent varieties are available in peaches, nectarines, and apples. The lower and interior fruiting branches of genetic dwarf trees, especially peaches and nectarines, low round pots tend to die quickly due to shading by the dense growth. However, the trees are small, so production of fruit on the extremities of the higher branches is not a serious problem as long as the branches are strong enough to hold the weight of the fruit. Pruning genetic dwarf trees mainly involves thinning the branches in the dormant season to open up the canopy and maintain the height and spread of the tree. Control tree size and strengthen limbs by removing branches at their point of attachment to the trunk or a larger branch , rather than by heading or “topping” them. For definitions of pruning terms, refer to the glossary.Full-sized trees on standard rootstocks can grow to 25 to 30 feet tall, while trees on semidwarfing rootstock can reach 15 to 20 feet tall. Both standard and semidwarf trees can be kept relatively small by pruning, but trees of this size may still grow too large for many backyard situations. An excellent selection of truly dwarfing apple rootstocks is usually available, and truly dwarfing rootstocks are being developed for most fruit species. Depending on the type of tree , full-sized and semidwarf trees may be trained to an open center, central leader, or fruit bush system.The open center, or vase-shaped, system is most commonly used on almond, apricot, cherry, fig, nectarine, peach, plum, and prune trees. Many pear, apple, and pistachio trees are also trained to this system. With this method, the center of the tree is kept free of large branches and vigorous upright shoots in order to allow sunlight to reach the lower fruiting wood. First growing season.

To create an open center tree, in about late April of the first growing season select three or four shoots that will become the primary scaffold branches and pinch back all other strong upright shoots to 4 to 6 inches long. When possible, these scaffold branches should be spaced several inches apart vertically and should be distributed evenly around the trunk, with the lowest branch about 18 to 24 inches above the ground. If growth is vigorous, the selected scaffold branches should be pinched back or headed to about 2 to 21⁄2 feet long in late May or early June to promote side branching and the development of secondary scaffold branches. Continue to pinch or head back unwanted branches but leave lateral shoots for next year’s fruit production. These unwanted branches will be removed later, but they provide shade for the trunk and main branches of young trees during the current growing season. If summer pruning was not done or was insufficient, create the open center during the dormant season. First dormant season after planting. Select three or four primary scaffold branches if this was not done the previous summer. Do not select scaffold limbs that grow directly above one another. Avoid upright limbs that are attached with narrow, acute angles because they tend to be weak at the point of attachment. Flat or horizontal limbs should be avoided as scaffold limbs, but they can be used if new shoots coming from them are directed upward and outward. For most species, a 45º angle for limb attachment is most desirable. If the tree grows poorly the first year, severely head back the primary scaffolds to three or four buds to promote vigorous growth the next year and correct the causes of the poor growth. Cherry, plum, and pear trees produce very upright growth. To promote tree spread, the scaffolds should be bent outward while still flexible or cut back to outside lateral branches. Other trees, such as apricots, peaches, and almonds, have a spreading growth habit and tend to produce lateral branches without heading. With these varieties it is often necessary to remove flatter-angled branches and leave more upright branches, thus maintaining the upward, outward growth pattern. Developing the mature tree. Heading or pinching the primary scaffold branches encourages secondary scaffold branches to grow from them. Allow two to three secondary branches to develop from each primary. Remove all other strong branches, preferably during the summer, to reduce competition with the scaffold branches and providesunlight to lateral fruiting branches and spurs. Head or pinch the secondary scaffold branches at 2 to 21⁄2 feet long to develop two to three “tertiary” branches from each secondary branch. Ideally, two branches should originate from each primary and secondary, and they should grow upward and outward but away from each other. However, shoot growth seldom conforms to this structure. Mature trees.

After 6 days and 15 days of storage, the kale disks showed lighter green coloration than the kale disks stored for 3 days . However, the kale leaf disks stored under constant light were lighter green than the kale disks stored under light/dark cycles and showed some brown or yellow discoloration after 3 and 6 days . By 15 days, the kale leaf disks stored under constant light lost nearly all green coloration and showed light and dark shades of browning with shape changes resulting from leaf folding and shrinkage . The kale leaf disks stored under constant darkness resembled those stored under constant light, except that the 3-day kale samples were darker green than the 3-day constant light-stored kale leaf disks , suggesting that the constant light may have constituted a greater stress on the kale leaves than constant darkness. These results indicate that post harvest storage with daily cycling of light and darkness improved the appearance of the kale leaf tissue compared to storage under either constant light or constant darkness. However, the preservation benefit obtained from post harvest storage under light/dark cycles at 22°C appeared to be less than that provided by refrigeration; kale leaf disks stored at 4°C with constant darkness, dutch bucket for tomatoes were comparable in their dark green coloration whether stored for 3, 6 or 15 days . Cabbage leaf disks stored under cycles of 12-hour light/ 12-hour darkness showed brown spots along the disk edges that increased in intensity over the storage period of 7, 14, and 21 days .

However, although the 7-day cabbage leaf samples were light green in coloration, the 14- and 21-day cabbage leaf disks stored under light/dark cycles had darker green coloration , suggesting increased photosynthetic activity over storage time. In contrast, although the cabbage leaf disks stored under constant light were also light green after 7 days of storage, the 14- and 21-day cabbage leaf disks were more yellow and included more brown discolorations . Remarkably, the absence of light exposure during post-harvest storage had a dramatic effect on the cabbage leaf disk coloration. Cabbage leaf disks stored under constant darkness at either 22°C or 4°C were pale tan or yellow after 3 days of storage . The constant darkness-exposed cabbage leaf disks stored at 22°C appeared nearly white in color by 14 and 21 days; those at 4°C had a yellowish appearance after 2 or 3 weeks of storage . Lettuce and spinach leaf disks tissue were nearly uniformly green, with little difference in color intensity between 3 and 6 days of storage under cycles of 12-hour light/12-hour darkness . By 9 days of storage under light/dark cycles, however, both lettuce and spinach leaf disks looked slightly less green, and most of the spinach leaf disks had distinct patches of yellow . In contrast, the loss of green coloration and increased yellowing over time was much more apparent in the lettuce and spinach leaf disks stored under constant light; the lettuce leaf disks were pale green by 9 days , and all the spinach disks had large yellow patches . Lettuce and spinach leaf disks stored under constant darkness displayed small brown patches by 3 days . After 6 and 9 days of storage under constant darkness, the lettuce disks had large wet patches of darkened tissue .

However, the spinach leaf disks stored under constant darkness at 22°C for 6 days completely disintegrated and therefore could not be moved for photographic imaging. Lettuce and spinach leaf disks stored in constant darkness at 4°C largely maintained dark green coloration at 6 days and were lighter green at 9 days, similar to that of the disks stored under light/dark cycles at 22°C . However, after 6 days of storage at 4°C, the lettuce leaf disks also displayed browning around the vascular tissues . Overall, the image analysis shown in Figure 1 suggests that post harvest storage under cycles of 12-hour light and 12-hour darkness may enable kale, cabbage, lettuce, and spinach leaf tissues to maintain physiological functioning for longer durations after harvest. The reduction in green color and appearance of brown discoloration suggests that post harvest storage in constant light or constant darkness may accelerate loss of tissue viability.To further characterize kale, cabbage, lettuce and spinach leaf health and viability during post harvest storage, we quantified chlorophyll content in leaf samples after storage under cycles of 12-hour light/12-hour darkness to leaf tissues stored under constant light or constant darkness. Three sets of comparative data are shown . Because our primary focus was to determine whether light/dark cycles were advantageous relative to constant light or constant dark storage conditions, we first conducted two-way comparative statistical analyses between data derived from the samples stored under light/dark cycles relative to comparable samples stored under the alternative condition. Figure 2 presents statistical analysis of storage-dependent differences in chlorophyll levels relative to dry weight at each time point. Additional file 1: Figure S3 shows similar analyses but of storage-dependent differences in chlorophyll levels relative to fresh weight. Finally, to evaluate whether there were significant changes in chlorophyll levels of each plant type over time, statistical analyses of differences in chlorophyll content at the beginning and end of the experiments for kale, cabbage, lettuce, and spinach are shown in Additional file 1: Figure S4.

Consistent with the loss of green coloration in the representative leaf disk samples shown in Figure 1A, post harvest storage of kale leaf disks in either constant light or constant dark led to significantly greater losses in kale chlorophyll content within 3 or 6 days of post harvest storage compared to storage under 12-hour light/12-hour dark cycles . Kale leaf disks stored for 15 days under constant light lost 97% and 93% of their original chlorophyll content relative to dry and fresh weight, respectively ; kale leaf disks stored under constant darkness lost 88% and 89% of their chlorophyll content relative to total dry and fresh weight, respectively . In contrast, 15 days of storage under cycles of 12-hour light/12-hour darkness led to loss of only 36% and 9% of the kale leaf disk chlorophyll relative to dry and fresh weight, respectively . Kale leaf disks stored at 4°C under constant darkness, however, performed statistically better than those stored under cycles of light/dark , with no significant decreases in chlorophyll content relative to total dry or fresh weight over the full 15 days of the experiment . Post harvest storage of cabbage leaf disks under light/ dark cycles resulted in significantly higher chlorophyll levels than storage under constant dark either at 22°C or 4°C at all time points examined . Indeed, cabbage leaf disks began with only modest chlorophyll levels . However, when the cabbage leaf disks were stored under either constant light or light/dark cycles, chlorophyll content increased over time with significantly higher levels remaining even after three weeks of storage . Light-induced synthesis is likely responsible for the elevated chlorophyll levels and the enhanced green coloration observed in the cabbage leaf images of the samples stored under light/dark cycles or constant light but absent in the cabbage leaf disks stored under constant darkness either at 22°C or 4°C . Storage under light/dark cycles was also more successful than constant light exposure in maintaining higher chlorophyll levels after long-term storage of 3 weeks . Over time, storage under constant light may be counterproductive; whereas cabbage leaf disks stored for 7 days under constant light had significantly higher chlorophyll content than leaf disks stored under light/dark cycles , by three weeks of storage, the leaf disks stored under light/dark cycles retained at least 2-fold more chlorophyll than samples stored under constant light . These results indicate that light during post harvest storage can have a profound effect on chlorophyll levels in cabbage, consistent with previous reports, blueberry grow pot and that diurnal cycling of light and darkness prolongs this benefit during longer term storage. Storage under cycles of 12-hour light/12-hour darkness also promoted chlorophyll retention in lettuce leaf disks, comparable to that of lettuce leaf disks stored under refrigeration; chlorophyll levels were statistically indistinguishable between lettuce leaf disks stored under light/dark cycles versus those refrigerated under constant darkness conditions after 3, 6 or 9 days of storage . Postharvest storage of lettuce leaf disks either at 22°C under light/dark cycles or under refrigeration resulted in no significant change in chlorophyll content over the course of the 9-day experiment, whereas the lettuce leaf disks stored under either constant light or constant darkness, lost more than 50% of their starting chlorophyll content . Chlorophyll content of spinach leaf disks was not significantly affected by treatment conditions for the first 3 days of post harvest storage . However, the spinach leaf disks stored at 22°C in constant darkness disintegrated by 6 days and were therefore unable to be further analyzed. Spinach leaf disks stored under light/dark cycles had similar chlorophyll content to those stored under constant light with relatively stable chlorophyll retention until day 9 when chlorophyll levels in both samples decreased significantly from initial levels . In contrast, refrigeration led to stable chlorophyll levels in the spinach leaf disks over the course of the experiment .

These results indicate that chlorophyll content of post harvest green leafy vegetables varies depending upon the storage conditions and suggests that storage under 12-hour cycles of light and darkness, known to maintain the plant circadian clock, can improve kale, cabbage and lettuce chlorophyll content maintenance relative to storage in constant light or constant dark. Perhaps surprisingly light/dark cycles during post harvest storage may be at least as beneficial as refrigeration with respect to chlorophyll content for cabbage and lettuce.Over time during post harvest storage, plant tissues typically show visible signs of tissue disintegration . To determine if maintaining light/dark cycles during storage of post harvest leafy vegetables could prolong tissue integrity, we compared electrolyte leakage from kale, cabbage, lettuce, and spinach leaf disks stored over time under cycles of 12-hour light/12-hour darkness to leaf disks stored under constant light or constant darkness at 22°C or constant darkness at 4°C. Figure 3 shows that post harvest storage under light/dark cycles and refrigeration were comparable, with respect to leaf tissue integrity maintenance of kale, cabbage and lettuce, as measured by electrolyte leakage. When directly comparing light/dark storage to other conditions, a statistically significant benefit to diurnal stimuli during storage was apparent relative to constant light for kale , constant darkness for cabbage and lettuce , and constant darkness and constant light for spinach . Post harvest storage under constant dark was detrimental to kale, cabbage, and lettuce tissue integrity, with at least 4-fold increases in electrolyte leakage, whereas storage under light/dark cycles at 22°C or refrigeration resulted in no significant increase in electrolyte leakage over the course of the experiment . Constant light treatment also led to significant increases in electrolyte leakage from kale and lettuce leaf disks, but not cabbage leaf disks, over the storage periods examined . Overall the results shown in Figure 3 and Additional file 1: Figure S5 provide evidence that daily cycles of light and darkness during post harvest storage resulted in superior leaf tissue integrity maintenance largely comparable to refrigeration, whereas either constant light or constant dark storage conditions were detrimental.Our results indicate that storage of kale, cabbage, lettuce, and spinach in light/dark cycles can improve the post harvest longevity of chlorophyll levels and tissue integrity. Next we were interested in determining whether plant maintenance under daily cycles of light and darkness affects human-health relevant metabolite content. In particular, we sought to examine whether kale and cabbage stored under light/dark cycles maintain their glucosinolate content longer than when stored under constant light, constant darkness, or refrigeration. Figure 4A shows total glucosinolate levels in kale leaf disks after 0, 3, 6 and 15 days of post harvest storage under different conditions. Individual glucosinolate levels are shown in Additional file 1: Figure S1. Total glucosinolate levels were comparable between kale leaf disks stored at 22°C under light/dark cycles and leaf disks stored at 4°C in the dark ; after 15 days of post harvest storage under these conditions, total glucosinolate levels decreased by less than 35% . In comparison to light/dark storage conditions, constant light or constant darkness exposure during storage resulted in significantly reduced glucosinolate content in the kale leaf disks . By 15 days of post harvest storage under constant light or constant darkness at 22°C, the kale leaf disks lost over 80% and 99% of initial levels, respectively.

IBtolerant cultivars belonging to the ‘Smooth Cayenne’ group develop symptoms at a slower rate than do the susceptible types, and the browning is primarily seen in the F region. Pineapples classified as IB-resistant show no TS or IB symptom but may display damage externally , also called “common chilling injury” .IB in pineapple is a result of the metabolic dysfunction from chilling-induced membrane damage . Loss of organellar structure permits the normally plastidic polyphenol oxidase access to phenolic compounds that are usually sequestered to the vacuole . The o-quinones intermediates produced by PPO act on the phenolic compounds, and are then converted to the polymers responsible for the internal browning associated with PCI . Chilling injury in pineapple is also associated with the production of hydrogen peroxide , which ages the cells . Therefore, higher PPO, phenolics, and H2O2 production are common biochemical markers of IB initiation . Although these metabolic changes have been well documented, our current understanding of chilling injury of pineapple is incomplete. This may be due in part to the lack of knowledge regarding the precise site of IB initiation in pineapple fruit. IB may occur at fruit vascular bundles in pineapple. This is plausible even though there are few existing data concerning pineapple fruit anatomy in relation to IB . However, data from tomato , mango , feijoa , vertical hydroponic nft system and especially banana and avocado support a relationship between VBs and IB. In avocado, fruit VBs separate and become stringy after seven weeks storage at 5.5 ◦C or four weeks storage at 0.5 ◦C .

The exudate leached from the chilling-damaged VB strands was presumed to cause tissue browning . In banana, after storage at 6 ◦C for 3 days, chilling-induced damage to fruit peel VBs was detected by histological staining . The ruptured VBs were also associated with blackening of the surrounding tissue . In pineapple fruit, the F/C region, where IB symptoms are initiated, is the VB-rich region. Thus, investigating the occurrence ofthe early events of IB development, i.e., TS atthe VBs of pineapple fruit, was the aim of this study. Four pineapple cultivars belonging to two groups, ‘Queen’ and ‘Smooth Cayenne’, with three levels of IB sensitivity were used to determine: if IB initiation, i.e., the appearance of translucency symptoms occurs randomly in any living fruit cell or specifi- cally at the VBs, if the VBs are the sites of biochemical activity associated with TS, and if different IB-sensitive cultivars show differences in VB structure and organization.The fruit were harvested at the mature green stage, approximately 18 weeks , or 21 weeks after floral induction. Fruit were harvested early in the morning and transported to the laboratory within 8 h. On arrival, the fruit were sorted by size and maturity. The uniformity of fruit maturation was determined by visual assessment of the outer exocarp, i.e., the opening stage of the fruit lets or eyes at the stem end of the fruit. Only fruit of which all the fruit lets were totally green were selected for further testing . At this stage, flesh color was light yellow, and soluble solids content was greater than 11◦Brix. Dirt and insects on the fruit were removed using an air blower. All leaves atthe stem end were removed and the peduncle was re-cut to remain one inch long. Forty-eight fruit of uniform maturity for each cultivar were selected for chilling injury determinations.

Fruit were either examined immediately after harvest or stored at 10 ◦C and 85% relative humidity for 7, 14, and 21 days to induce translucency symptoms or chilling injury , or stored at 25 ◦C continuously for the same period .The PCI markers, H2O2 and PPO, were not detected by stereomicroscopic analysis of non-chilled fruit , whereas the presence of phenolic compounds was observed . After the TST pineapple fruit was exposed to 10 ◦C for two weeks, TS were observed on approximately 9% of the total surface area of the fruit. These spots only appeared in the F/C, notin the C or the F regions. Tissues with these localized translucent areas at the F/C region were carefully sectioned and further investigated. Higher contents of phenolic compounds, PPO activity, and H2O2 were detected in the translucent tissue as the development of an intense brown color using our diagnostic histological staining assays . These areas of TS served as an indicator of IB initiation and were further examined under a light microscopy with higher magnification . It was clearly evident that the coloration was more concentrated in the translucent tissue . Furthermore, PPO activity and H2O2 production in the F/C tissue were not detected in non-VB tissue . In this paper, we used a non-specific stain as an indicator of PPO activity, which in turn acted as a proxy for IB. Our assay does not allow us to definitely detect PPO activity, but instead, may be a good indicator of the spatio-temporal occurrence of IB. The staining appears only in chilled TST fruit and is absent from tissues with no manifestation of IB, i.e., non-chilled and senescing tissue . Furthermore, no staining was visible in MD2, a cultivar with no known PPO increase after chilling, and no IB symptoms . These data indicate that for the purpose of this study, the catechol staining was an adequate marker for IB.

VB anatomy at the F/Cregionof control, senesced, and chilled tissue of the IB-susceptible TST pineapple fruit were further investigated under a scanning electron microscope . The VBs consisted of xylem elements, phloem, and VB caps of sclerenchyma fiber. The xylem elements were composed of lignified cells and occupied the center of the VBs. The phloem consisted of thin-walled cells that were small in size and were located adjacent to the xylem elements. A zone of small parenchymal cells surrounding the phloem tissues was modified into sclerenchyma fiber . Such cells were less abundant or lacking around the VBs, resulting in the small size of the VBs in the F region . There were no obvious differences in the non-VB parenchymal cells taken from the F/C region in control compared to chilled fruit . However, there was damage at the phloem tissue in chilled fruit, which resulted in the collapse and flattening of these areas . This region was examined in detail. Unlike chilled fruit, neither TS nor IB was present in senesced fruit , and the tissues of the VB in non-chilled and senesced fruit were similarly healthy with no damage to the phloem in any of the sections examined . There was a high co-occurrence of TS and phloem collapse, nft hydroponic system regardless of tissue location . In most cases, the xylem elements and sclerenchyma fiber around the VB were unaffected by chilling. The specificity of phloem deterioration was clear:it occurred only in chilled fruit, neither in non-chilled nor in senesced tissue. The early changes in phloem integrity that precede the appearance of TS in cold-stored TST were investigated. In this experiment, TST pineapple fruit kept at 10 ◦C for 21 days showed 10% TS, whereas the fruit stored for less than 21 days did not show TS . Then, the degree of phloem damage during this chilled period was categorized as follows: “None Detectable” A), “Mild,” i.e., loss of phloem cell integrity but no collapse B–C), and “Severe,” i.e., collapse of at least 50% of the phloem tissue D). The result showed that, at day 0 and in the senesced tissue , only 25% of all VBs showed mild damage of the phloem. After 7 days at 10 ◦C, the percentage of VBs that showed mild damage increased from 25 to 90%, with the remainder starting to show severe damage. After 14 days storage, severe phloem damage increased from 10 to 40%, even though no TS were observed. After 21 days at 10 ◦C, TS were visible at the base of the fruit and, in this region, all VB showed phloem damage with more than 70% classified as severe. In addition, when the non-affected tissue adjacent to the TS region was observed, a similar proportion of phloem collapse was found . These results clearly suggest that phloem collapse occurs before TS, and that the phloem is the site of chilling injury.Identifying the site of IB occurrence due to low temperature storage might help to better understand the mechanism of PCI in pineapple fruit and other similarly affected species. The present study uniquely demonstrates that IB in pineapple occurred at the phloem and different IB-sensitive cultivars showed differences in VB structure and organization. Similar results were obtained when the entire experiment was repeated 2–3 times.The current results clearly showed that the VB tissue of pineapple fruit was the site of H2O2 accumulation.

There were also higher levels of phenolic compounds and PPO activity specifically due to chilling stress . In addition, phloem damage was found only in the chilling-stress-induced translucent tissue . There may be three explanations as to why TS selectively occurs at the phloem and why phloem collapse leads to TS. The first involves phloem properties,the second, phloem ultrastructure, and the third, changes in cell wall degradation of phloem tissue.The cells in the phloem tissue may contain different cellular properties, e.g., membrane composition or a different antioxidant system, compared to the cells in other tissues. To our knowledge, the cellular properties of phloem tissue have not yet been studied. However, some data supporting this idea was found in Paspalum dilatum Poir. After 3 days at 10 ◦C, ultrastructural change of the chloroplasts in the phloem parenchyma preceded that of the chloroplasts in the lower mesophyll. This different rate of change was also associated with the severity of damage of the upper mesophyll .The phloem tissue contains sieve elements, companion cells, parenchyma cells, and in some cases includes: fibers, sclereids, and laticifers . Transportation through the sieve tube occurs by pores in the sieve plates or sieve areas between the sieve tube elements. These pores allow the plasma membrane of a sieve tube element to be continuous with that of its neighboring sieve tube element. Chilling induces the loss of cell turgor, vacuolization of the cytoplasm, and swelling disintegration of cell organelles . This will lead to leakage of the substrate needed by PPO for the synthesis of the brown substances as proposed by Woolf et al. for avocado. These browning-related substances could be conducted throughout the fruit via the continuous sieve tube connection. The collapse of the phloem would lead to the exudates from the VBs filling up the intercellular air spaces of the surrounding tissue, resulting in translucent spots , and eventually, browning, as proposed for other species .After 21 days at 10 ◦C, paradoxically,the non-TS and adjacentTS regions had a similar proportion of phloem collapse. This may be explained by variation in the number of layers of sclerenchyma cells at the lateral side of the VBs in these regions. Non-TS regions have more fiber layers, which effectively restrict phloem exudates to the surrounding parenchyma tissue, preventing the intercellular air spaces from filling with liquid. The opposite would be true for the TS-regions, which have fewer layers, allowing for a greater flow of exudates to the intercellular spaces and the rapid appearance of TS. Only after a longer period of chilling stress, would the protective function of the sclerenchyma fibers in the non-TS tissue become less effective, leading eventually to TS.Degradation of the cell wall might play a role in phloem collapse upon IB induction. The walls are non-lignified and are weaker in structure compared to the tracheary elements of the xylem . Each sieve tube element is interconnected with the metabolically active companion cells by plasmodesmata, which act as cytoplasmic bridges . In addition, around the plasmodesmata, there is a deposition of callose . As maturation of sieve element progresses, the callose and the middle lamella cell wall material in this region are degraded to form a plate structure with widened pores . In citrus, chilling causes pitting and browning of the flavedo and induces the activity of  -1, 3-glucanases . The capability of this enzyme in degrading callose, which is deposited in the plasmodesmata in sieve element, might play a role in the phloem collapse upon chilling in pineapple. Alternatively, it is possible that the non-VB parenchymal cells located next to the VB tissue might be the sites of IB initiation .

Water used for blanching, post-blanching cooling, and cooking of fruits and vegetables can, in general, be collected and reused for the initial washing of incoming products without treatment . Reuse of flume water. Instead of discharging flume water to the wastewater stream, it can be recovered, filtered, and reused continuously in fluming applications. Alternatively, flume water can be recovered and recycled for use in equipment pre-rinsing and pre-soaking applications elsewhere in the facility . Reuse of compressor cooling water. Cooling water from compressors can be reused as seal water in vacuum pumps instead of fresh water, or as secondary water for other purposes, such as equipment pre-soaking . Warm cooling water can also be stored in insulated tanks for later use in facility cleaning, pre-soaking, and equipment pre-rinsing applications .The U.S. fruit and vegetable processing industry spent nearly $810 million on purchased fuels and electricity in 2002, making energy a significant cost driver for the industry. Energy efficiency improvement is an important way to reduce these costs and to increase predictable earnings in the face of ongoing energy price volatility. Considering the negative impacts of the 2001 spike in U.S. natural gas prices on the industry’s operating costs, stackable planters as well as more recent sharp increases in natural gas prices across the nation, energy efficiency improvements are needed today more than ever.

Many companies in the U.S. fruit and vegetable processing industry have already accepted the challenge to improve their energy efficiency in the face of high energy costs and have begun to reap the rewards of energy efficiency investments. This Energy Guide has summarized a large number of energy-efficient technologies and practices that are proven, cost-effective, and available for implementation today. Energy efficiency improvement opportunities have been discussed that are applicable at the component, process, facility, and organizational levels. Preliminary estimates of savings in energy and energy-related costs have been provided for many energy efficiency measures, based on case study data from real-world industrial applications. Additionally, typical investment payback periods and references to further information in the technical literature have been provided, when available. A key first step in any energy improvement initiative is to establish a focused and strategic energy management program, as depicted in Figure 6.1, which will help to identify and implement energy efficiency measures and practices across and organization and ensure continuous improvement. Tables 5.1 to 5.3 summarize the energy efficiency measures presented in this Energy Guide. While the expected savings associated with some of the individual measures in Tables 5.1 to 5.3 may be relatively small, the cumulative effect of these measures across an entire plant may potentially be quite large. Many of the measures in Tables 5.1 to 5.3 have relatively short payback periods and are therefore attractive economic investments on their own merit.

The degree of implementation of these measures will vary by plant and end use; continuous evaluation of these measures will help to identify further cost savings in ongoing energy management programs. In recognition of the importance of water as a resource in the U.S. fruit and vegetable processing industry, as well as its rising costs, this Energy Guide has also provided information on basic, proven measures for improving plant-level water efficiency, which are summarized in Table 5.4. For all energy and water efficiency measures presented in this Energy Guide, individual plants should pursue further research on the economics of the measures, as well as on the applicability of different measures to their own unique production practices, in order to assess the feasibility of measure implementation.This work was supported by the Climate Protection Partnerships Division of the U.S. Environmental Protection Agency as part of its ENERGY STAR program through the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. Many people within and outside of the fruit and vegetable processing industry provided valuable insights in the preparation of this Energy Guide. The authors are particularly grateful to the following people for their helpful comments and advice during the development of this Energy Guide: John Batura of Campbell’s Soup Company, Joseph Benevides of Ocean Spray Cranberries, Austin H. Bonnett , Alan Christie of the J.R. Simplot Company, Elizabeth Dutrow of the U.S. Environmental Protection Agency, Dan Fonner of Heinz North America, Paul Halberstadt of ConAgra Foods, Al Halvorsen of Frito-Lay, Don Hertkorn of ICF Consulting, Ted Jones and Ilene Mason of the Consortium for Energy Efficiency, John Malinowski of Baldor Electric Company, Aimee McKane of Lawrence Berkeley National Laboratory, Leland McPherrin of Escalon Premier Brands, Rob Neenan of the California League of Food Processors, Richard Pate of the Pacific Gas and Electric Company, Linda Raynes of the Electrical Apparatus Service Association, Jon Russett of General Mills, and Terry Young of ConAgra Foods.

Any remaining errors in this Energy Guide are the responsibility of the authors. The views expressed in this Energy Guide do not necessarily reflect those of the U.S. Environmental Protection Agency, the U.S. Department of Energy, or the U.S. Government. We discovered that there are only a few categories of interest in a highly domesticated breeding population and that a small number of features are that ordinal shape categories are highly heritable and that the features needed for accurate classification are also heritable.The data released with this article contain digital images of 6,874 strawberry fruit from 572 hybrids originating from the University of California, Davis, Strawberry Breeding Program. The data for this article, including pre-processed images , processed images , and extracted features , are available on Zenodo. The pre-processed images typically contained multiple berries per image along with a data matrix bar code indicating the genotype ID and other elements of the experiment design. The processed images are 1,000 × 1,000 pixels-scaled binary images of individual fruit. The extracted features data set is provided as a CSV file. Additionally, snapshots of the code and data supporting this work are available in the GigaScience repository, GigaDB. We hope that the release of these data assists others in developing novel morphometric approaches to better understand the genetic, developmental, and environmental control of fruit shape in strawberry, and more broadly in other fruits, vegetables, and specialty crops.k-Means clustering rapidly detects patterns in large, multidimensional data sets used for clustering, decision making, and dimension reduction. It is an iterative algorithm that partitions a data set into a pre-defined number of non-overlapping clusters, k, by minimizing the sum of squared distances from each data point to the cluster centroid. A centroid corresponds to the mean of all points assigned to the cluster. Here, we used k-means to cluster flattened binary images . Individual fruits were segmented from the image background as a binary mask, normalized by the major axis, resized to 100 × 100 pixels, and flattened into a vector . We represented each image as a 10,000-element vector containing binary pixel values. We were able to rapidly and reliably assign images to classes using k-means clustering. In this experiment, we allowed k, the number of permitted categories, to range from 2 to 10. This range was chosen because we anticipate that a human-based classification system would not have the speed or reliability needed for this task, particularly for larger values of k.As high-throughput phenotyping for external fruit characteristics becomes of interest to specialty crop researchers, we expect that this work will have various applications in both applied and basic plant research, stacking pots intellectual property protection and documentation, and waste reduction. Our study showed that strawberry fruit shapes could be robustly quantified and accurately classified from digital images. Most importantly, our analyses yielded quantitative phenotypic variables that describe fruit shape , arise from continuous distributions, and are moderately to highly heritable . We accomplished this by translating 2D, digital images of fruit into categorical and continuous phenotypic variables using unsupervised machine learning and morphometrics. We found that mathematical approaches developed for human face recognition were powerful for strawberry fruit shape phenotyping , that unsupervised shape clustering was robust to sample size deviations, and that only a few quantitative features are needed to accurately classify shapes from images , indicating a paradigm appropriate for genetic dissection.

Digital plant phenotyping is able to empower quantitative genetic analyses by providing heritable and biologically relevant, latent phenotypes in a cost-effective manner. In many cases, these latent traits are derived from PCA, multidimensional scaling , structured equation modeling , persistent homology , or auto-encoding convolutional neural networks, which can be exceedingly abstract and difficult to interpret biologically but may also reveal unexpected patterns of phenotypic and genetic variation. Many of the features described in this study, along with those reported by Turner et al. and Gage et al. , had high heritability and are exciting targets for future quantitative genetic analyses, including GWAS and genomic prediction, which have been shown to be successful for shape features in recent work in rice, apple, and pear. However, the H2 of 1 selected feature in this study, EigenFruitPC3, was estimated to be 0.00. Similar results were reported in carrot for pixel-based root and shoot features, apple for elliptical Fourier leaf shape features , and corn for pixel-based shoot features. Turner et al. attributed the null H2 of root shape characteristics to low phenotypic variation between the inbred parents and genotype × environment interactions. This pattern, while seemingly present, was not discussed in detail by either Migicovsy et al. or Gage et al.. While there may be many drivers for this pattern, we hypothesize that the null estimate may arise from the pixel-based descriptors describing more complex aspects of fruit or root shape. If the non-genetic component of a multivariate phenotype is large, then performing PCA on that multivariate trait could produce leading PCs that describe mostly non-genetic variance . However, there are too few reports to adequately determine the likelihood and causal source of this phenomenon. We empirically derived the shape progression produced in the present study through the application of a new method, PPKC, and used these mathematical categories to interpret the extracted shape features . Ordinal categorical traits are commonplace in quantitative genetic studies, a current standard for phenotyping external fruit characteristics, and enable understanding and explanation of complex, latent space plant phenotypes . PPKC specifically considers the relationship between a cluster at k and all clusters for values <k as a covariance matrix and projects this k-dimensional space to 1 dimension using eigen decomposition. Ordination using dimension reduction techniques, including PCA, correspondence analysis, and MDS has been previously proposed and used in community ecology. Theoretically, the eigen decomposition step of PPKC could be replaced with another technique. However, unlike methods using eigen decomposition, which progressively subdivides variation such that the position on the leading axis is fixed regard-less of the number of axes examined, the position of samples on MDS axes may change when different dimensions are extracted, making MDS axes arbitrary and without meaning other than a convenient reference. PPKC identified 4 exemplary strawberry shape categories in the population that we studied, which were characterized by a progression from ”longer-than wide” to ”wider-than-long”. This ordinal scale can be used in breeding and research programs as traits of interest, or it can be used to organize and interpret more abstract quantitative features, such as EigenFruitPCs or SEM latent variables, through supervised machine learning algorithms. Critically, this gradient agreed with the arbitrarily defined progressions in previous reports. However, unlike previous studies, which suggested using 9 ordinal or11 nominal shape categories, our work presented empirical evidence for a smaller number of mathematically defined shape categories. We determined that k = 4 was the appropriate level of complexity on the basis of the visual appearance of the discovered clusters , high H2 estimates , and the information criteria calculated for the k-means models. Interestingly, PPKC can determine a visually, reasonable phenotypic gradient up to k = 8 despite strong evidence of overfitting for k > 4. We extrapolate that PPKC should continue to work beyond k = 9 so long as new clusters are distinct and do not arise as an artifact of overfitting k. The specific genetic factors that give rise to variation in fruit shape in octoploid, garden strawberry are currently unclear or understudied. The selective pressure exerted on fruit shape in strawberry could have affected large-effect loci, in which case ordinal phenotypic scores are likely to be sufficient for identifying genetic factors affecting fruit shape. Loss- and gain-of function mutations have played an essential role in identifying genes affecting fruit shape in tomato, a model that has been highly instructive and important for understanding the genetics of fruit shape and enlargement in plants.

The U.S. EPA’s ENERGY STAR Building Upgrade Manual recommends a stepwise approach to recommissioning, in which a series of strategically-ordered building “tune up” strategies are pursued in order. First, lighting and supplemental loads should be assessed, then the building envelope, then controls, then testing, adjusting and balancing, then heat exchange equipment, and finally heating and cooling systems. Most of these steps relate to HVAC system components or factors that will directly affect HVAC system energy consumption . For more information, the U.S. EPA’s ENERGY STAR Building Upgrade Manual should be consulted . Energy monitoring and control systems. An energy monitoring and control system supports the efficient operation of HVAC systems by monitoring, controlling, and tracking system energy consumption. Such systems continuously manage and optimize HVAC system energy consumption while also providing building engineers and energy managers with a valuable diagnostic tool for tracking energy consumption and identifying potential HVAC system problems. Several industrial case studies from the United States indicate that the average payback period for HVAC control systems is about 1.3 years .Non-production hours set-back temperatures. Setting back building temperatures during periods of non-use, such as weekends or non-production times, nft channel can lead to significant savings in HVAC energy consumption. Duct leakage repair. 

Duct leakage can waste significant amounts of energy in HVAC systems. Measures for reducing duct leakage include installing duct insulation and performing regular duct inspection and maintenance, including ongoing leak detection and repair. According to studies by Lawrence Berkeley National Laboratory, repairing duct leaks in industrial and commercial spaces could reduce HVAC energy consumption by up to 30% . One commercial building in Apple Valley, California, adopted a technique called the mobile aerosol-sealant injection system to reduce duct leakage. The application of MASIS resulted in a reduction in overall duct leakage from 582 cfm to 74 cfm, leading to a 34% increase in the overall efficiency of the building’s HVAC system . Variable-air-volume systems. Variable-air-volume systems adjust the rate of air flow into a room or space based on the current air flow requirements of that room or space. Variableair-volume systems therefore work to more closely match HVAC load to heating and cooling demands, which reduces energy use. Adjustable-speed drives . Adjustable speed drives can be installed on variable volume air handlers, as well as recirculation fans, to match the flow and pressure requirements of air handling systems precisely. Energy consumed by fans can be lowered considerably since they are not constantly running at full speed. Adjustable-speed drives can also be used on chiller pumps and water systems pumps to minimize power consumption based on system demand.

Heat recovery systems. Heat recovery systems reduce the energy required to heat or cool facility intake air by harnessing the thermal energy of the facility’s exhaust air. Common heat recovery systems include heat recovery wheels, heat pipes, and run-around loops. The efficiency of heat pipes is in the 45% to 65% range , while the efficiency of run-around loops can be slightly higher, in the 55% to 65% range . Fan modification. Changing the size or shape of the sheaves of a fan can help to optimize fan efficiency and airflow, thereby reducing energy consumption. In a case study from the automotive industry, a Toyota plant optimized the sheaves of its fans in lieu of installing ASDs on fans. Toyota found better savings and payback periods with sheave modification than they anticipated to experience from ASDs . Efficient exhaust fans. Exhaust fans are standard components in any HVAC system. Mixed flow impeller exhaust fans offer an efficient alternative to traditional centrifugal exhaust fans. Mixed flow impeller fans are typically 25% more efficient than centrifugal fans, and can also be cheaper to install and maintain. The expected payback period for this measure is around two years . Use of ventilation fans. Ventilation fans installed in the ceilings of work areas can help destratify the workspace air, leading to better circulation of cool air in summer and warm air in winter, and more even distributions of temperature from floor to ceiling. Such fans can help to reduce the load on building heating systems by helping to “push down” warm air that rises to the ceiling during facility heating months.

Yasama Corporation U.S.A., a manufacturer of soy sauce, installed new high bay ceiling fans to improve air circulation at its Salem, Oregon, facility in 2004. Previously, to provide heat during the winter, the company operated ceiling-mounted heaters with 15 hp fans in its production area. However, the fans didn’t de-stratify the air in the production area’s tall ceilings, nor take advantage of the heat given off by process equipment. Furthermore, to provide ventilation in the summer, the company ran the heater fans in “fan only” mode in conjunction with six 3 hp exhaust fans to remove hot air. The new high-bay ceiling fans were operated using only 1.5 hp motors, which were expected to lead to electrical energy savings of 48,000 kWh per year and electricity cost savings of $2,500 . Furthermore, the company expected to save significant amounts of natural gas in heating months through reduced operation of the heaters. Cooling water recovery. If available, secondary cooling water from municipal sources can be leveraged to reduce chiller energy consumption. In Washington, Boeing partnered with Puget Sound Power and Light and the King County Department of Metropolitan Services to recycle secondary treated cooling water into its chiller system. By doing so, Boeing reduced its water consumption by 48 million gallons per year, leading to projected savings of 20% in its cooling energy consumption . As an additional benefit, Boeing also expected to save on refrigerants and treatment chemicals for its cooling tower water. Solar air heating. Solar air heating systems, such as Solarwall® , use conventional steel siding painted black to absorb solar radiation for insulation. Fresh air enters the bottom of the panels where it is heated as it passes over the warm absorber. Fans distribute the air. Using this technology, Ford Motor Company’s Chicago Stamping plant turned the south wall of its plant into a huge solar collector . Energy savings were estimated to be over $300,000 per year compared to conventional gas air systems. Capital costs were $863,000 resulting in a payback period of less than three years. In addition to energy savings, the system was said to provide clean fresh air for employees, even out hot and cold spots in the plant, and reduce emissions. However, this measure is only of interest for buildings in cold climates, and the potential benefits should be analyzed based on the local conditions of each site. Building reflection. Use of a reflective coating on the roof of buildings in sunny, hot climates can save on air conditioning costs inside. Two medical offices in Northern California used reflective roofs on their buildings; one reduced air conditioning demand by 8%, hydroponic nft the other reduced air conditioning demand by 12% . For colder climates, heat lost due to cool roofs also needs to be taken into account, and often negates savings. In addition to location and weather, other primary factors influence energy savings, such as roof insulation, air conditioning efficiency, and building age. Reflective roof materials are available in different forms and colors. Roof gardens on a flat roof improve the insulation of buildings against both hot and cold by providing both heat and air conditioning . In winter, green roofs can freeze, so they carry a slight heating penalty but often still yield net energy savings . In addition, a roof garden can increase the lifetime of the roof, provide and reduce runoff, and reduce air pollution and dust. Today, Germany installs over 10 million ft2 of green roofs a year, helped in part by economic incentives . The Gap Headquarters in San Bruno installed green roofs in 1997 . In addition to saving energy and lasting longer than traditional roofs, a roof garden absorbs rain, slowing run-off to local storm drains. Other simple options for decreasing building HVAC energy use exist for certain conditions. Shade trees reduce cooling for hot climates. Shade trees should be deciduous trees and planted on the west and southwest sides of the building . Trees planted on the north side of the building in cold climates can reduce heating in winter by shielding the building from the wind. Vines can provide both shade and wind shielding. Building insulation. 

Adding insulation to a facility will nearly always result in the reduction of utility bills. Older buildings are likely to use more energy than newer ones, leading to very high heating and air conditioning bills. Even for a new building, adding insulation may save enough through reduced utility bills to pay for itself within a few years . Various states have regulations and guidelines for building insulation, for example, California’s Energy Efficiency Standards for Residential and Nonresidential Buildings . Going beyond regulated insulation levels may be economically beneficial and should be considered as part of the design of a new building, as well as for reconstruction of existing buildings. For refrigerated warehouses, much higher levels of insulation are preferred. Low emittance windows. Low emittance windows are another effective strategy for improving building insulation. Low emittance windows can lower the heat transmitted into a building and therefore increase its insulating ability. There are two types of Low-E glass, high solar transmitting and low solar transmitting . The U.S. DOE supports the development of new window and glazing technology, while ENERGY STAR provides a selection of rated Low-E windows. New window and glazing technology is being developed continuously around the world.22Turning off lights in unoccupied areas. An easy and effective measure is to encourage personnel to turn off lights in unoccupied building spaces. An energy management program that aims to improve the awareness of personnel with regard to energy use can help staff get in the habit of switching off lights and other equipment when not in use. Lighting controls. Lights can be shut off during non-working hours by automatic controls, such as occupancy sensors that turn off lights when a space becomes unoccupied. Occupancy sensors can save up to 10% to 20% of facility lighting energy use . Numerous case studies throughout the United States suggest that the average payback period for occupancy sensors is approximately 1 year . In a case study from the pharmaceutical industry, at the Merck office and storage building in Rahway, New Jersey, lighting panels were programmed to turn off automatically during expected periods of building non-use . Annual savings amounted to 1,310 MBtu per year, which corresponded to avoided energy-related carbon dioxide emissions of nearly 260 tons per year . Manual controls can be used in conjunction with automatic controls to save additional energy in smaller areas. Other lighting controls include daylight controls for indoor and outdoor lights, which adjust the intensity of electrical lighting based on the availability of daylight. An example of energy-efficient lighting control is illustrated by Figure 11.1, which depicts five rows of overhead lights in a workspace. During the brightest part of the day, ample daylight is provided by the window and thus only row C would need to be turned on. At times when daylight levels drop, all B rows would be turned on and row C would be turned off. Only at night or on very dark days would it be necessary to have both rows A and B turned on . These methods can also be used as a control strategy on a retrofit by adapting the luminaries already present. Exit signs. Energy costs can be reduced by switching from incandescent lamps to light emitting diodes or radium strips in exit sign lighting. An incandescent exit sign uses about 40 W, while LED signs may use only about 4W to 8 W, reducing electricity use by 80% to 90%. A 1998 Lighting Research Center survey found that about 80% of exit signs being sold use LEDs . The lifetime of an LED exit sign is about 10 years, compared to 1 year for incandescent signs, which can reduce exit sign maintenance costs considerably. In addition to exit signs, LEDs are increasingly being used for path marking and emergency way finding systems. Their long life and cool operation allows them to be embedded in plastic materials, which makes them well suited for such applications .