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 ,cannabis grow lights 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.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 of these 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. A sustained rise in ethylene production upon warming after chilling is characteristic many chilling sensitive tissue . MRI provided data that allowed the spatial resolution of the internal changes in chilling-induced fruit that were not available using conventional methods. ADC maps of an equatorial section of a tomato are shown in Fig. 1a. These heat maps indicate changes in water mobility, which increased during and after chilling; presumably because of chilling-induced disintegration of membrane integrity. After chilling at 0 ◦C for one week, the amount of voxels in the entire fruit in the yellow-red range increased compared with that of the non-chilled fruit . This general trend continued during two weeks of cold storage .
However, when these fruit were transferred to 20 ◦C, the number of yellow-red voxels throughout the slice decreased in the pericarp, but less so in the center of the fruit. Quantitative data better illustrate differences among tissues . When compared with the pericarp,cannabis grow tent the inner tissues showed higher signal frequencies at greater ADCs after 2 weeks refrigeration followed by room temperature storage relative to the controls . D-values were calculated for the whole fruit in order to make comparisons with ethylene and respiration data obtained from whole fruit. These values were higher in one week-chilled fruit compared with other storage periods . After two weeks at 0 ◦C and with further storage at 20 ◦C, the D-values were similar to the control . An increase after 1 week of chilling was also observed in carbon dioxide, ethylene evolution, and ion leakage . However, while these criteria remained higher than those in the non-chilled control fruit, the D-values declined relative to those in the control . The mean D-values of distinct spatial regions of the fruit were then calculated. No changes from the control were recorded in the pericarp after chilling for 1 or 2 weeks at 0 ◦C . However, after an additional week of storage at 20 ◦C, D-values decreased to levels observed in the non-chilled control. This may be indicative of the ability of marginally chilled tissue to repair the chilling-induced physiological damage that accumulated during chilling when warmed to non-chilling temperatures . There was little correspondence between changes in these D-values and changes in membrane ion leakage values; which were both derived from pericarp tissue . Ion leakage increased after cold exposure while Dvalues did not. There was some variability in pericarp ADC-values as mentioned earlier; cold-stored fruit varied compared to those reconditioned at 20 ◦C . Even when percent membrane permeability in the pericarp was considered over shorter time intervals, e.g., 15, 25 min, etc., changes due to chilling were still asynchronous with pericarp D-values . For the interior columella and locular tissues, the D-values increased after 1 week of chilling and remained at this elevated level for the remainder of the storage period . This response was significantly different from that of the pericarp. When the controls were compared to fruit kept in the cold for 1 and 2 weeks, D-values for interior tissue correlated reasonably well with changes in respiration and ethylene production . However the inner fruit D-values showed an identical pattern to changes in ion leakage, which is a destructive assessment of pericarp changes . This suggests two possibilities: that each individual assay acts as a proxy for similar underlying biological processes, but that they each encapsulate some divergent mechanisms, or that in response to cold, the pericarp and inner fruit may be modulated in different temporal time frames with varying amplitudes.Membrane disintegration and the subsequent physiological changes in chilled fruit may promote higher water mobility detected as increased D-values. Interpretation of these data suggest that the sensitivity and responsiveness to chilling differs between the pericarp and core tissues. This may arise due to the physical distance from the epidermis to the fruit center. An endogenous temperature gradient may be imposed across the fruit creating an inherent delay in the response and any subsequent adaptation to cold, between pericarp and core tissues. Although this possibility cannot be ruled out, the relatively small diameter of Micro-Tom fruit should minimize this effect.
Of more importance may be the heterogeneous nature of the fruit tissues. Differences in their physiochemical properties and functionalities could produce different biological outcomes to chilling. The realization that fruit responses to cold are asynchronous should inform our view when designing and interpreting data from experiments to study chilling injury. The aim of the experiments described here was to explore basic fruit post harvest biology using MRI and thereby gain new insight into CI.While not fast enough for commercial use,the MRI scan time of 27 min were still much faster than the more than 3 h needed to measure respiration, ethylene evolution and ion leakage. In addition to being faster, MRI also provides spatially resolved data. A long-term goal would be to refine this technology to enable its practical application as an economical, robust and rapid on-line detector of post harvest disorders in packinghouses or even along the supply chain. This would allow each stakeholder to have better control over produce quality. Examining the response of different cultivars that vary in fruit size, pericarp thickness, ratio of columella to locular tissues, and sugar to acid content, and to a variety of low temperature in cubations will also be necessary to facilitate MRI’s adoption in commercial settings.Tomato is the world’s second largest vegetable crop rich in nutrients. Tomato fruit development includes three stages. The first stage is characterized by an increase in cell number and starch accumulation, followed by cell enlargement with starch degradation and soluble sugar accumulation in the second stage. Fruit ripening is the last stage, associated with the accumulation of soluble sugars, carotenoids, organic acids, and volatile organic compounds in fruits. The chlorophyll accumulation and photosynthetic activity of green fruits influence the nutritional components and flavor of ripening tomato fruits. Some genes have been reported to affect chlorophyll accumulation, chloroplast development and fruit quality. As negative regulators, DE-ETIOLATED 1/high pigment 2 and UV-DAMAGED DNA-BINDING PROTEIN 1/ high pigment 1 are involved in chloroplast formation and chlorophyll accumulation in tomato fruits. The tomato GOLDEN2-LIKE transcription factors SlGLK1 and SlGLK2 play an important role in chloroplast formation and chlorophyll accumulation.Evidence suggests that the SlGLK2 gene is predominantly expressed in fruits and that the latitudinal gradient of SlGLK2 expression influences the production of unevenly colored tomato fruits. Over expression of the APRR2- LIKE gene, the closest homolog of SlGLK2, increased the size and number of chloroplasts and enhanced chlorophyll accumulation in green tomato fruits.