Changes in expression at the onset of ripening occurred earlier in ED than in controls. Previous work in our laboratory also demonstrated that ED advanced the onset of ripening, apparent from an earlier onset of color development and corresponding induction of flavonoid genes, and accelerated accumulation of anthocyanins and sugars after the onset of ripening . Other studies examining the effects of water deficit have not found a temporal advancement in the onset of ripening, but did find that water deficits accelerated anthocyanin accumulation after the onset of ripening . That some studies have not found a temporal advancement in the onset of ripening may reflect differences in the nature of the applied water defi- cits. ED fruit in the current study was subject to water deficit for more than a month prior to the onset of ripening, while fruit in Castellarin et al. and Deluc et al. was subject to water deficit for only 1–2 weeks prior to the onset of ripening. Alternatively, drainage pot the weekly sampling interval used in Castellarin et al. and Deluc et al. may have missed the acceleration as the onset of ripening occurs over *7 days.
Regardless, these studies show that differences in fruit composition brought about by water deficit are established in a very short period of time surrounding the onset of ripening whether through advancement in the onset of ripening, accelerated accumulation, or both. At this time, there is no direct evidence demonstrating that changes in ripening brought about by water deficit are indeed mediated by ABA. However, several studies demonstrate a strong correlation between the timing of the onset of ripening and ABA. In this study, berry culture treatments demonstrate that ABA, in the presence of high sugar, can bring about many of the changes associated with onset of ripening in grape. Wheeler et al. brought about an advancement in the onset of ripening through the repeated application of ABA to immature field-grown Cabernet Sauvignon, and Deluc et al. demonstrated that the acceleration in ripening in response to water deficit was correlated with increases in ABA. Without more sophisticated genetic studies, the evidence for ABA’s role in mediating the effects of water deficit on fruit development remains corollary.Several lines of evidence suggest that changes in berries cultured with exogenous sucrose and ABA parallel changes in field-grown fruit at the onset of ripening. Softening, color development, and changes in gene expression associated with onset of ripening in were brought about by culture on high sucrose ? ABA, but not with low or high sucrose treatments alone. The 2 and 10% sucrose concentrations used in this study approximate sugar concentrations of berries prior to and at , the onset of ripening in the field . ABA concentrations in the culture media match or exceed peak ABA concentrations at the onset of ripening in the field .
The induction of color development in the high sucrose ? ABA treatments was not surprising given the strong evidence for sugar and ABA induction of anthocyanin biosynthesis both in model systems and in grape cell culture . Cultured berries grew and increased Brix at a rate similar to field-grown berries for the range of Brix represented by 2–10% sucrose. Similarly, the elasticity of both field-grown and cultured berries decreased from approximately 6 to 1 MPa or less from 61 to 84 DAA . The parallels in patterns of gene expression are striking. Those genes strongly up-regulated at the onset of ripening in control and ED field-grown fruit were similarly regulated between high sucrose, and high sucrose ? ABA treatments . Other genes, such as VvSUT2-1 were up-regulated at the onset of ripening in the field, while in culture they were up-regulated in response to sugar only . Furthermore, other genes constitutively expressed during ripening in field experiments exhibit the same pattern of expression in culture . Koyama et al. also demonstrated that, in culture, exogenous ABA brings about changes in gene expression analogous to the onset in ripening in the field. Decrease in elasticity in response to exogenous ABA suggests that ABA treatment resulted in decreases in mesocarp cell turgor, although turgor was not measured directly in this study. The decrease in elasticity in culture is attributable to a decrease in the DWs between the symplast and apoplast , and/or a decrease in cell wall rigidity through loosening of the cell wall, and not to desiccation because the berries grew during culture. In fruit, there is evidence that ABA plays a role in both of these processes through the stimulation of various invertases and cell wall-modifying enzymes . The large weight gain, swelling of the mesocarp tissue, and berry cracking in low sucrose ? ABA treatments, supports a role for ABA in stimulating cell wall loosening. This could be an interesting focus of future study. In the introduction, we discussed the idea that components of regulatory networks can be conserved across disparate developmental processes that share commonalities.
Sugar and ABA signals act across diverse processes . Assuming that sugar and ABA signals work to control similar physiological processes in these different contexts what common physiology underlies their action? One strong candidate is the control of turgor, which is intimately connected to germination , stomatal control, the onset of ripening , and the control of growth.Exposure of susceptible plant tissues to non-freezing temperatures below 10–12 ◦C induces a physiological disorder called Chilling Injury . There appears to be two phases in the development of CI. The first phase is initiated in the cold and could involve a change in membrane fluidity or enzyme activity . Overt symptoms develop after prolonged chilling or upon warming to non-chilling temperatures . These secondary symptoms are predicated by primordial events initiated in the cold, and include a host of metabolic and physiological changes that include increased membrane permeability , increased respiration and ethylene production , uneven ripening, disease susceptibility, water soaking and surface pitting . A technique that could detect the earliest physiological changes associated with CI would foster a better understanding of the initial events leading to this disorder, and point to more effective ameliorative action. Magnetic resonance imaging is a nondestructive imaging technique, which is increasingly used to visualize and quantify fruit physiological response to endogenous or exogenous stimuli . MRI uses the magnetic properties of nuclei and their interactions with radio frequency and applied magnetic fields to produce an image . Variations in the chemical composition and integrity of cellular structures can change the movement of water within and among tissues. These changes can be detected as modifications in the relaxation times of the protons in water, drainage planter pot which in turn alters the signals used to construct MR images . Diffusion-weighted MRI of tissues provides a quantitative measure of the apparent diffusion coefficient of water, instead of estimations of water mobility from relaxation measurement that include the influence of translational mobility, composition and other factors . In addition, a spatially resolved map of the apparent diffusion coefficient of water can be obtained, which could help to understand and quantify the development of disorders such as CI within the tissue. MRI has been used to gain insight into early phases of different post harvest physiological disorders before the manifestation of external symptoms . These include core breakdown in pear water core disorder , internal browning and mealiness in apple . There are few reports where MRI was used to detect the early stages of CI in sensitive produce. In persimmon, MR images of cold-stored fruit were distinct from those stored at ambient temperature . In zucchini squash , MRI provided enough data to act as a predictor of where water soaking would occur in the epidermis after the cold-storage.
These studies both indicated that MRI has great potential for studying CI in fruit tissues. Tomato is one of the most important horticultural crops both economically and as a genomics, molecular, biochemical, and physiological model for biological processes occurring in fleshy fruits . Like most subtropical fruit, tomato is susceptible to CI. Studies with tomato fruit could leverage existing functional genomics resources to pinpoint the molecular basis of this trait. To our knowledge, MRI has not been used to study CI in this species. We used the dwarf cultivar ‘Micro-Tom’ because it is the functional genomics model for tomato . Its high-density growth, short life cycle and concentrated fruit-set makes it possible to obtain harvests of 500 fruit or more per square meter per year . Because tomato post harvest studies can be hampered by biological variability , the availability of numerous, similarly aged fruit makes Micro-Tom a convenient experimental model for post harvest studies . Furthermore, we have previously characterized Micro-Tom fruit physiological response to a range of post harvest chilling temperature-time combinations , and used this information to design a metabolomics investigation of CI. This has established a baseline with this cultivar for the further CI studies we exploit here. The specific objective of this study was to determine if MRI could detect some of the earliest physiological changes that accompany CI in tomato fruit. Current methods of assessing the occurrence and severity of CI are: time consuming , destructive , or occur only after the activation of secondary, downstream events . These methods are time-proven and are indispensable, but there is a need for non-destructive methods with equivalent or better sensitivity to those currently used. MRI potentially offers such advantages and could be an important complementary tool for studying incipient CI. We show that MRI can provide spatio-temporal resolution of chilling induced changes in MicroTom tomato fruit prior to development of downstream symptoms.Chilled fruit were removed from 0 ◦C and allowed to slowly warm to 20 ◦C before CO2 and ethylene production were measured. Twelve fruit were evaluated per treatment by placing four fruit in each of three 500 mL glass containers. These jars were sealed for 1 h and a 1-mL sample of the head space was withdrawn using a syringe and its CO2 concentration was measured with an infrared gas analyzer as previously described . Ethylene production was measured from a 2.5 mL sample of the head-space using a Gas Chromatograph equipped with a flame ionization detector. These two samples were taken within 30 s of each other from the same jar.Each fruit was cut into four radial segments, cleaned of adhering locular tissue, washed for 5 s in running tap water, blotted dry, and one segment was placed in each sector of a 4-sectored Petri dish under aseptic conditions. The dishes were placed in plastic tubs lined with wet paper towels and loosely covered with aluminum foil. The tubs were held at 12.5 ◦C for 18 h to produce ‘aged’ tissue, i.e., to allow the tissue to overcome the wound-induced alterations in membrane permeability . After transferring to room temperature for 1 h, the four aged segments from each Petri dish were put into a 50 mL plastic centrifuge tube containing 20 mL of an aqueous solution of 0.2 M mannitol. Preliminary experiments determined that 0.2 M was isotonic for these excised radial segments The conductivity of the bathing solution was measured with an Extech Model 480 digital conductivity meter every 5 min for 30 min and then less frequently for 180 min with gently shaken between readings. After 3 h the tubes were capped, frozen at −20 ◦C and warmed to room temperature and frozen and thawed twice before the total conductivity of the solution was measured at room temperature after 1 h of shaking. Ion leakage was expressed as percent of total and plotted over time. The linear increase in ion leakage from 0.5 to 2.0 h was used to calculate the rate of ion leakage .The aim of this study was to examine the extent to which MRI could detect changes in chilled Micro-Tom tomato fruit. Based on our previous data using Micro-Tom , fruit were stored at 0 ◦C in the dark for 0 , 1, or 2 weeks, and the fruit that were chilled for 2 weeks were held for 1 week at 20 ◦C. The onset of CI was evaluated using changes in respiration, ethylene evolution and ion leakage . One week of chilling increased all ofthese indicators to levels that were maintained during an additional week of cold-storage . In fruit transferred to 20 ◦C after 2 weeks of chilling, the rate of respiration returned to that of the pre-chilling levels, ion leakage remained at the same elevated levels and ethylene production continued to increase.