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, 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, plant pot with drainage 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 ofthe 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. 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 incubations will also be necessary to facilitate MRI’s adoption in commercial settings. Most of what we currently know about how plants cope with low temperatures stems from the work carried out in the temperate model plant Arabidopsis, where it has been studied in vegetative tissues in relation to cold acclimation, a process integrated with developmental programs that results in extensive transcriptome and metabolome reorganization which appears to act, at least in part, to protect membranes and proteins against the severe dehydration stress that occurs during freezing. By using mostly seedlings, the regulatory factors influencing the expression of cold regulated genes have been identified. Three cold induced transcriptional regulatory factors known as C-repeat binding factor control the expression of a major regulon of COR genes to confer plant freezing tolerance, and may play a role in chronic low temperature adaptation. Upstream of the CBF regulatory hub, two cold-sensing pathways have been described. One involves ICE1. The other involves calcium and the calmodulin binding transcription activators CAMTA3 and CAMTA1. In addition, some important components mediating cold and freezing tolerance through CBF-independent pathways has been described. Besides transcriptional regulation, there are evidences which indicate that cold acclimation is also regulated at the chromatin, post-transcriptional, translational and posttranslational levels. Further, ABA-independent and -dependent pathways regulate cold-responsive genes, and ABA acts synergistically with the cold signal. Although much attention has been paid to ABA in relation to the cold response, pot with drainage holes there is growing evidence that other hormones such as auxins, brassinosteroids, ethylene, jasmonic acid and salicylic acid are involved in cold acclimation. In general, basic cold responses can be shared among different plant species and organs, although, some structural and regulatory differences have been observed between tolerant and sensitive plant species. In fruits, however, cold might have an impact on a subset of specific characteristics and eventually affect ripening. Apple and some pear cultivars require cold acclimation to set up ripening. In apple, a CBF like gene promotes softening in absence of ethylene and, probably, cold and ethylene act independently and synergistically with each other to induce fruit softening. Little is known about low temperature responses in summer fruits such as peach because the chilling period occurs naturally as winter cold comes, when plants have not yet fruits. The horticultural industry uses similar temperatures to those triggering cold acclimation to preserve fruit quality after harvest. Despite widespread use, this technology has its restrictions, as many fruits and vegetables are sensitive to low temperatures and develop a syndrome named chilling injury. Peach fruits subjected to long cold storage periods can develop a form of CI called mealiness/woolliness, a flesh textural disorder characterized by a lack of juiciness, which appear only after fruits have shelf ripened at room temperature. Peach exhibits a high degree of genetic variability for chilling tolerance, with the most sensitive cultivars being damaged after 1 week of cold storage and the most tolerant remaining undamaged for at least 5 weeks. Genetic analysis indicates that chilling injury in peach is a quantitative trait, and a number of QTLs for chilling injury have been mapped to the peach genome.
Most of the reports on mealiness emphasize the changes in the cell wall during shelf life ripening after cold storage. Recently, large-scale approaches have identified new peach genes associated to mealiness during shelf life. Nevertheless, the information about what happens during cold storage is scant. During cold storage physiological alterations has been described. Firmness and ethylene production were reduced. Alterations in cell wall transcriptome, enzyme activity and in cell wall polymers metabolism still occur during cold storage, which affect in the manner fruits ripened during subsequent shelf life. Further, stress responsive genes increase during cold storage while genes related to energy metabolism decrease. Unfortunately, these reports did not go deep in the analysis of the genes and their functions and, failed to associate gene expression to chilling sensitivity as they were based in the response of a single genotype subjected or no cold. Contrasting genotypes can serve as a powerful tool for understanding the physiological and molecular mechanisms of chilling tolerance in peach. In a preliminary study the expression of ten cold induced genes was associated to the tolerance to chilling injury. More recenty, Dagar et al. identified a group of differentially expressed genes between two varieties at harvest, which are probably related to their tolerance or susceptibility to develop CI. In this study, we have used an adaptation for the gene expression data of the Bulked Segregation Strategy; dubbed herein as the Bulked Segregant Gene Expression Analysis . We used the custom cDNA Chill peach microarray as expression profiling platform on RNA from pools of fruits from siblings of the Pop-DG population exhibiting extreme cold responses. Our approach was validated and extended to a number of individual members of the population with different degrees of cold susceptibility by using medium throughput Fludigm RT PCR.Siblings from Pop-DG mapping population, segregating for chilling injury, were used in this study. Mesocarp samples from fruits of the following Pop-DG siblings were used: 49/59, 84/85, 86/87 and 132/133 with high sensitivity to mealiness and 71/ 72, 88/89, 134/135, 142/143 with low sensitivity . These Pop-DG siblings with similar horticultural characteristics but with extreme differences on mealiness development were selected because their sensitivity phenotype was consistent for 3 years prior this study . In all cases, fruits were harvested at the mature commercial stage according to Kader & Mitchel with flesh firmness of 12–14 lb, soluble solid content of 11–14% and tritrable acidity of 0.5–0.7% . A group of 12 fruits M were directly allowed to ripen at 20uC to the edible firmness of 2–3 lb as controls. For cold treatments, M fruits were forced-air cooled at 0–2uC within 6 h of harvest and were then stored at 5uC with 90% relative humidity for 1, 2 and 3 weeks . Chilling injury of each sibling after the cold storage period was expressed as Mealiness index , i.e the proportion of measured fruits with mealiness when ripened for 2–3 days at 20uC. Mealiness was assessed as the percentage of free juice content accordingly to Campos-Vargas et al. using the quantitative method described by Crisosto et al.. Fruits shelf ripened after one week of cold storage were checked for other chilling disorders as in Martinez-Garcia et al.. The samples representing at least 6 fruits from each genotype and treatment were bulked and immediately frozen in liquid nitrogen before storing at 280uC until they were used for RNA isolation.For the microarray experiments, equal amounts of RNA from each genotype in a given control or treatment group were mixed in the corresponding S and LS pools. The RNA pools were all hybridized using the ChillPeach microarray. All samples were compared using a dye-swap design against a common superpool reference, composed of equal amounts of RNA obtained from all the mesocarp samples. Three replicates from each sample pool were hybridized in each case, one of them dye-swapped. RNA purification, sample preparation and hybridization to Chillpeah microarray were performed as described in Ogundiwin et al.. Intensity values were obtained as the median of ratios using GenePix 4000B scanner .