br Materials and methods br Results
Materials and methods
Discussion In general, CI symptoms are often visible only after the chilled products have been removed from coolstore to a warmer temperature (Schirra and Cohen, 1999). However, in the present work, \'satsuma’ mandarin fruit stored at 2°C showed chilling symptoms after 4 weeks storage, and the development of chilling symptoms increased with storage time. Some HWD treatments applied in this study-delayed onset of CI development, particularly the 47.5°C for 2 and 5 and 50°C for 2min treatments which reduced CI incidence and severity after 8 weeks storage (Fig. 1). This supports previous findings on ‘Fortune’ mandarins (Schirra and D’hallewin, 1997) which showed a protective effect of hot water (50–52°C for 3min) against CI. One of the most noticeable changes observed in stored citrus fruit is the accumulation of ethanol and acetaldehyde, which increase during storage (Echeverria and Valich, 1988). After 4 weeks, the amount of Anti-cancer Compound Library kinase products was enhanced concomitant with occurrence of CI development at 8 weeks storage, supporting the finding of Schirra (1992) that there can be a good relationship between amount of CI and amount of anaerobic products (Table 2). Ethylene evolution and respiration rate of various heat-treated fruit was significantly affected by storage period (Table 2). After 4 weeks storage at 2°C, ethylene evolution and respiration rate was correlated with CI development in peel. The results were similar at 8 weeks, but there was a decline in ethylene evolution with increasing peel damage (Table 2). Increased respiration rate and ethylene production may indicate CI even in apparently sound fruit (Schirra, 1992) and, the occurrence of CI may be followed by an early increase in ethylene and respiration rate (Ren et al., 1999). This has been confirmed here with treatments which reduced CI effectively suppressing ethylene evolution and the rise in respiration rate (Table 2). Exposing plants to moderate heat treatments causes weak oxidative stress that modulates antioxidant levels and induces tolerance to a subsequent severe stress (Li, 2003). Storage of fruit at 2°C for 8 weeks decreased catalase (CAT) activity significantly, while peroxidase (POX) activity increased slightly (Table 2). Overall, the treatments that sustained high CAT activity and suppressed POX elevation during storage were protective treatments against CI (Table 3). Elevated levels of CAT in hot water treated mandarins showed suppressed CI. Generally, activity of antioxidant enzymes increased when the oxidant level increased with environmental stress (Ren et al., 1999). Therefore, the higher activity POX in peel may be linked to the higher cell damage as a response to stress (Li, 2003). In the work reported here, high activity of POX in most treatments, but especially at 55°C for 5min was associated with severe peel damage. The present work has also focused on the effects of heat treatments followed by low temperature storage on the specific gene expression of the V-PPase and V-ATPase in the peel and the pulp since these enzymes may be important indicators of tonoplast integrity and important to the maintenance of fruit acidity. The 5-fold induction of both V-PPase and V-ATPase at 8 weeks for the 55°C treatment seems to be a consistent response to the highest heat treatment (Table 5). It is likely that the loss of tonoplast integrity due to damage induces compensation by induction of tonoplast proteins. Of interest was the rapid decline in V-PPase and induction of V-ATPase exhibited in juice sacs with heat treatments, and the recovery to levels similar to the control after 8 weeks cold storage in treatments which had minimum damage (HWD50). This contrasts with the high levels of transcript exhibited in treatments which had heat damage (HWD55). Vacuolar H-ATPase is a primary active pump located at the vacuolar membrane (tonoplast) of plant cell (Sze, 1985). Because of the importance of the V-ATPase in plant vacuole, it can be expected that the activity of the V-ATPase be modulated to cope with environmental and metabolic changes (Dietz et al., 2001). One day after heat treatment, V-ATPase transcript in peel was down-regulated in comparison to the control (Table 4). It has previously been reported that V-ATPase activity is unaffected by a chilling treatment (4°C; Canel et al., 1995) but in our case, there was a rapid decline (1 day) in transcript level which was partially restored at 8 weeks .There was, however, evidence of a decline in transcript level for V-ATPase in HWD at 55°C for 2min (Table 4). The decrease in transcript in the peel is correlated to the level of damage in response to either heat treatments or chilling stress (Table 4). The higher levels of transcript at 8 weeks in the more successful treatments provide some evidence that the V-PPase and the V-ATPase may be instrumental in protecting against chilling damage by sustaining energization of the vacuolar membrane.