Tomato suffers from PCI: storage at 0–12 °C followed by returning the fruit to room temperature can lead to a range of symptoms, varying from mild e.g. lack of favor and poor texture, to severe i.e. development of surface lesions, discoloration, accelerated softening, failure of fruit to ripen and higher susceptibility to post harvest decay. Numerous studies in tomato fruit, including those on PCI, have traditionally focused on only one tissue, the pericarp. Since PCI is a complex phenomenon, the lack of robust, practical solutions could be a consequence of the use of fragmentary approaches when analyzing the progression of the disorder. Te pericarp and the central tissue, the columella, account for most of the fruit fresh mass in round tomatoes, however, in cherry tomato the pericarp is comparatively thinner and the placenta and locular tissue are significantly larger, and contribute to most of what is eaten. Chilling can affect internal tissues and there is abundant evidence describing the differential development of physiological and biochemical processes in the different tomato fruit fractions. It is also known that PCI targets processes occurring across different biological levels and time frames. The precise order of events triggered by PCI is unknown, pot with drainage holes but one of the primary events is the production of reactive oxygen species.

If cold exposure is mild or limited, cellular homeostasis and fruit quality will be maintained through the activation of alternative oxidases, and protective proteins such as dehydrins, in part, by the regulation of upstream factors of the cold response pathways such as C-Binding Repeat transcription Factors. Beyond this threshold, or during rewarming of previously cold-stored fruit, progressive loss of selective membrane permeability due to lipid peroxidation may occur. This in turn, can lead to secondary metabolic and physiological dysfunction such as the leakage of water, solutes and metabolites, ROS accumulation, bursts in ethylene and respiratory rates, and later, ripening disruption, surface lesions and fungal infestation. Because of the functional specialization of fruit tissues, and, the many processes that constitute PCI, the progression of this disorder would be expected to also differ across tissues. Therefore, the aim of this work was to examine the components of the molecular, biophysical, biochemical and physiological processes affected by PCI in both the pericarp and columella over short and long-term cold-storage. This could allow us to build a more holistic and integrated view of this phenomenon.Carbon dioxide and ethylene production are standard biomarkers for PCI in tomato fruit. Respiration supplies the cell with energy, and ethylene is a ubiquitous plant hormone involved in stress response, senescence and fruit ripening. In climacteric fruit, the rates of respiration and ethylene production increase with the onset of ripening. In this study, 2.5 °C, 5 °C and 12.5 °C were used as chilling temperatures, with 2.5 °C and 5 °C expected to induce PCI while 12.5 °C should not and acts as a cold-storage control.

The production of these gases was suppressed during the period of chilling and increased up to 100% relative to the control after rewarming to 20 °C , which was proportional to the occurrence of PCI. Ethylene production behaved similarly under chilling, but unlike respiration where there was a characteristic burst of CO2 within 1day of rewarming, the occurrence of the ethylene peak was delayed . Tis was consistent with a previous study where breaker fruit stored at 3 °C for 3 weeks displayed a peak in this gas after 3 days at 20 °C.Chilling often leads to poor fruit color development, surface pitting and decay after rewarming, the extent of which is proportional to the severity of the cold stress. The CII encapsulates these data and is expressed as a score from one to four. The rewarmed fruit, especially those stored at 2.5 °C, failed to ripen normally , had pitted surfaces , and evidence of decay , as evidenced by higher CII values, compared to those stored at control temperature . Fruit hue angle values were also assayed, as this is a quantitative and reliable indicator of tomato fruit color changes due to PCI. Hue angle values decreased as redness increased, a trend only seen in control fruit . It confirmed that chilling at both 2.5 and 5 °C adversely affected color development from the first week of cold storage and that rewarming could not reverse these alterations. Chlorophyll degradation, and carotenoid and lycopene accumulation are responsible for red color formation and are inhibited by cold in tomato fruit.

Structural and conformational changes of cellular membranes are amongst the first physiological events induced by PCI. These alterations reduce membrane selective permeability leading to electrolyte leakage. However, we observed no increase in this parameter over time, under 2.5 °C-or 12.5 °C storage, or even when 2.5 °C-stored fruit was rewarmed . At 5 °C, results were not linear since there was a decrease in ion leakage followed by an increase after rewarming . We only recorded ripening-, rather than chilling-induced membrane damage when control fruit was rewarmed. Ion leakage was therefore not an accurate biomarker for PCI in this experiment, and our data supports the view that it is highly variable and dependent on pre- and post harvest conditions.Magnetic resonance imaging is a valuable technique for the non-invasive monitoring of tissue physiological status. Diffusion weighted MRI allows the mapping of intra-tissue water mobility, in the form of apparent difusion coefficient images . ADC are hypothesized to change due to PCI from chilling-induced biochemical and physiological alterations such as membrane leakage. Non-invasive and simultaneous assessment of the changes in water mobility patterns in the columella, locules and pericarp under chilling and control conditions would be valuable in developing a holistic view of the development of PCI. Overall, the MRI data illustrated that chilling silences key physiological processes that occur during ripening , and that the pericarp, columella and the locules could be clearly resolved based on their water mobility profles in response to temperature. The pericarp remained unresponsive for the duration of the experiment under 2.5 °C, including rewarming, while the columella and locules were more variable. The locular tissue was the least dynamic fraction under both chilling and control temperatures, and the columella was more responsive after 3 weeks of storage and after rewarming .D-values increased mostly, and to a greater magnitude in control fruit, and in chilled fruit after rewarming , which correlates with our ion leakage data . Ripening-associated liquefaction after the transfer to room temperature likely contributed to increased water mobility. Likewise, the only changes in chilled fruit were recorded after prolonged storage or after rewarming . This could be attributed to chilling-induced damage since fruit were ripening-inhibited and started to manifest PCI symptoms as revealed by the CII data . Cumulative chilling injury likely compromised the tissue’s capacity to undergo normal ripening. Interestingly, both the ion leakage and ADC data illustrate phenomena associated with water mobility and membrane permeability. While both parameters varied under rewarming after control storage, only ADC appears to be chilling-responsive. The MRI highlights the need to examine each tissue to characterize PCI’s progression and symptomatology, since the most studied fraction, the pericarp, large pot with drainage may not reflect processes occurring in the whole fruit. MRI showed that the inner fruit tissues, although traditionally not well-studied, undergo chilling injury. This was further underscored by the higher incidence of seed browning in chilled fruit compared to control fruit , after rewarming . PCI-induced seed browning was also shown in eggplant and pepper. Browning is due to the production of melatonin from chilling-induced increases in polyphenol oxidase activity , its phenolic substrate, and importantly, membrane decompartmentalization, which facilitates PPO access to phenols and the production of brown pigments.Cold induces lipid peroxidation of cellular membranes, with MDA as a byproduct. MDA production is considered a biomarker for PCI-induced loss of membrane integrity in tomato fruit both during cold-storage, and after rewarming.

MDA production in the pericarp and columella was significantly different across time points , with the greatest changes occurring after rewarming preceded by at least 2 weeks of chilling. MDA content peaked earlier in the columella compared to the pericarp . These are signs of a differential response to oxidative stress in both fractions, and that MDA content of the external and internal fruit tissues followed independent programs.D-values and MDA contents report phenomena related to changes in membrane integrity due to PCI, but from different perspectives. D-values indicate tissue water mobility status that might result from membrane disruption, while MDA indicates oxidative degradation of the membrane. Interestingly, these parameters showed opposite trends: the pericarp was cold-unresponsive for D-values at 2.5 °C but was variable in terms of MDA production. Conversely, the columella fuctuated more in terms of D-values under cold, but for MDA production it changed less than the pericarp .Starch is the primary storage compound in green tomato fruit. During post harvest storage of breaker fruit, sugar accumulation will depend on starch degradation, and exogenous factors affecting its breakdown will influence quality. Starch content was higher in columella compared to the pericarp across experimental conditions , in agreement with previous studies. Interestingly, both chilling and ripening reduced starch content. Ripening caused more drastic changes, however, starch degradation was still active under cold. This contrasts with the trend seen in other metabolic parameters, i.e., respiration and ethylene production , which were suppressed during chilling. Starch decline was largest after the first week of chilled storage, with a 48.8% and 62.7% decrease in the pericarp and columella, respectively. When tissues stored for 3 weeks were contrasted with those stored for the same time, but followed by rewarming, starch in the columella decreased under both control and chilling conditions, but only under control conditions in the pericarp. This suggests that after 3 weeks of cold storage, starch degradation in the pericarp reached an irreversible plateau, similar to reports in banana; while starch breakdown was still responsive to rewarming after chilling in the columella. Total fruit starch content was negatively correlated with respiration and ethylene evolution rates at 2.5 °C. Sugars produced from starch degradation during the cold likely fueled metabolic processes after rewarming through increased respiration. Starch may therefore be an important biomarker of early post harvest chilling injury.Changes in the transcriptional abundance of key genes likely occur in ‘waves’ and are among the earliest triggers of the plant cold response. Five genes were selected based on their known connection to ripening, changes in redox balance, and cold response. They included genes involved in ethylene biosynthesis and in cold, dehydration, and oxidative stress responses. Their relative abundance was quantified after 1 and 24 hours chilling to investigate rapid changes during short-term storage, and after 3 weeks to investigate changes after prolonged storage. Correlative analyses of the expression patterns between these genes were performed. Significant correlations may indicate coordinately regulated processes, and it was of interest to determine if they were altered by cold treatment. Ethylene biosynthesis. In tomato, the products of 1-aminocyclopropane-1-carboxylic acid synthase isoform 2 , and ACC oxidaseisoform 1 , encode key enzymes in ethylene biosynthesis. They are both expressed at elevated levels during climacteric and post climacteric ethylene production. Te expression of ACO1 and ACS2 in both pericarp and columella was influenced by the length of storage, but only under control conditions , consistent with their role in fruit ripening . ACS2, was also expressed during post-climacteric ripening in another study. Chilling altered the patterns of gene expression over time. For ACO1, transcripts levels were steady in both fractions pre- and post-rewarming, even though ethylene increased 26-fold after rewarming . Down-regulation of ACO1 by cold was reported by others, and in our study, was especially apparent in the columella after 3 weeks of cold storage . ACS2 expression varied only under conditions that promoted ripening and was more prominent in the columella . With ACO1, there were no differences in expression between the pericarp and columella fractions under either chilling or control conditions, whereas ACS2 expression varied spatially under the control temperature. This emphasizes that the expression of both genes followed different spatial dynamics. Both genes were co-expressed at 12.5 °C , however chilling attenuated this strong correlation. Cold stress response. The C-Repeat Binding Factor 1 transcription factor is a master regulator of plant cold stress response, and some studies show that its expression is also induced in tomato fruit during post harvest cold-storage.