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 .