IW reduces CI – does it influence the ‘primary cause’ or only delay expression of

Chilling injury can be considered as consisting of two processes – primary and secondary events. The primary event is the initial, rapid response to the chilling temperature; this stage is reversible. Sustained primary damage causes a cascade of secondary effects after a period of time. It seems obvious that IW does not influence the initial events (which may begin within minutes of chilling) but it may delay the onset of secondary events (i.e. delaying the onset of visible symptoms) and prevent secondary events from becoming irreversible (thereby allowing the tissue to ripen normally after cold storage). There have been a number of attempts to explain the biochemical and physiological mechanisms underpinning these primary and secondary events.

Changes in lipid composition of membranes (decrease in phospholipids, decrease in unsaturated fatty acids) cause a decrease of fluidity that makes the membrane dysfunctional and decreases functionality of proteins associated with it (Lurie et al., 1997; Staehelin and Newcomb, 2000). Therefore, membranes that contain a higher degree of unsaturated fatty acids (linoleic and linolenic acid) are more chilling resistant (Lee et al., 2005; Zhang and Tian, 2009) than chilling sensitive plants that usually contain much a higher proportions of saturated fatty acids (Murata and Nishida, 1990). Increased concentrations of unsaturated fatty acids were reported in heat-treated tomatoes compared to control (Whitaker, 1994), and it is possible that the heated fruit had more fluid membranes (Lurie et al., 1997). It is suggested that IW mimics heat treatment in influencing lipid composition in the cellular membranes. Since intermittent warming could maintain high level of phospholipids and increase degree of unsaturation of fatty acids

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concentrations during a rapid readjustment of metabolism (Wang, 1982), IW probably helps the membrane to return the lipids to a less gel-forming condition and undoes the primary response.

One of the earliest theories related to the cause of CI proposed that accumulation of toxic metabolites was caused by a temperature induced imbalance in metabolism (Nelson, 1926 as cited in Saltveit and Morris, 1990) and the inability of chilling sensitive plants to detoxify these compounds at low temperatures (Caldwell, 1990). Subsequent warming of fruit apparently induced higher metabolic activity that removed excess intermediates and replenished deficiencies that developed during chilling (Wang, 1993). For instance, reduction of superficial scald in cool-stored apple by intermittent warming could be associated with either inhibition of toxic compounds such as α-farnesene and its oxidation products or enhanced catabolism of conjugated trienes during warming periods (Alwan and Watkins, 1999; Rudell et al., 2011). Since IW of fruit induced higher metabolic activity in each warming occasion (Figure 6.6 and Figure 6.11), it has the potential to reverse toxic metabolites and undo the primary response.

While IW may have direct effect on removing toxic metabolites build up during chilling by inducing higher metabolic activity, it may have an indirect effect of quenching other toxic compounds such as ROS by enhancing antioxidant activities and subsequently reducing CI. Several enzymes are involved in scavenging free radicals in plant defence systems. Different postharvest treatments (e.g. heat treatments) are able to manipulate antioxidant systems in tissues of fruit and vegetables (Soto-Zamora et al., 2005). Wang and Baker (1979) reported that IW increased the contents of polyamines (spermidine and spermine) and stimulated activities of free radical scavenging enzymes in cucumbers and sweet peppers. In the present study, IW enhanced red colour development (Figure 4.2); Tonucci et al. (1995) found that red colouration in tomato was highly correlated with lycopene content, a potent quencher of ROS (Di Mascio et al., 1989). Tomatoes develop red colour as chloroplasts change to chromoplasts. Two main electron transport systems in plant cells are located in chloroplasts and mitochondria and usually responsible for the generation of oxygen free radicals during chilling stresses (Purvis and Shewfelt, 1993). If the chloroplast is a source of ROS and the chromoplast is not, then it is possible that reduction of CI by IW in the present study was through stimulating an antioxidant protection mechanism that suppressed chilling-induced oxidative stress and quenched ROS.

171 Synthesis of HSPs during different heat treatments is well documented in the literature and many researchers attributed acquisition of low temperature tolerance to induction of these HSPs (Lafuente et al., 1991; Sabehat et al., 1996). However, no information could be found in the literature about synthesis of HSPs in response to intermittent warming. Alleviation of CI and induced chilling tolerance by heat treatments usually involve exposure of plant tissues to a relatively higher temperature than the temperature employed during intermittent warming. Therefore, it is not clear if IW is perceived as a heat treatment by the plant cells. Lafuente et al. (1991) indicated that exposure of plant tissues to a sudden jump of temperature about 5-10 °C above the normal growing temperature may well be enough to induce the synthesis of HSPs. Therefore, it is possible that warming the fruit intermittently during low temperature storage may stimulate synthesis of HSPs that could be involved in recovery from chilling stress and confer protection from subsequent chilling stress. This is a specific and testable hypothesis which has not been pursued in this thesis and needs to be investigated in future work.

Overall, it is possible that IW can repair the primary damage and protect the tissue against subsequent low temperature. IW could reverse phase changes of the membranes, allow repair of damaged membranes and organelles, restore metabolic imbalances and allow metabolism of toxic metabolites. Periodic warming of tissues could maintain high levels of phospholipids, increase degree of unsaturation of fatty acids, increase the concentration of spermidine and spermine, and stimulate activities of free radical scavenging enzymes. Heat treatment induces HSPs, suppresses oxidative activity, and maintains membrane stability. All of these processes may contribute to the beneficial role of IW in enhancing chilling tolerance of tissues.

7.5. Reduction of CI by IW has both ethylene dependent and independent