The cascade of physiological events leading to chilling injury : the effect of post-harvest hot water and molybdenum applications to lemon (citrus limon) fruit.
New emerging markets such as Japan and the United States require cold sterilisation of South African citrus fruit as a phytosanitary standard against fruit fly. However, citrus fruit are chilling susceptible, with lemons being the second-most chilling susceptible after grapefruit. Chilling injury is a physiological rind disorder; the occurrence of which is despite its prevalence in horticultural commodities, not well understood. Therefore, the aim of this study was to investigate physiological compounds regulating chilling susceptibility or resistance in citrus fruit, with special emphasis on lemons. Furthermore, the potential of hot water dips or “molybdenum soaks” to maintain a certain level of physiological compounds which determine manifestation of chilling injury symptoms in citrus fruit was investigated. Moreover, it was attempted to create an understanding of the order in which physiological compounds mitigate chilling injury. Lemon fruit from different farms known to be chilling susceptible or resistant were obtained during the 2007 and 2008 harvest season. Thereafter, fruit were treated by soaking for 30 min in 1μM NaMo04.2H20 solution followed by a 2 min HWD 47 or 53°C. Treated fruit were waxed, weighed and stored at -0.5°C for up to 28 days and sampled for chilling injury evaluation 7, 14, 21, or 28 days into cold storage. A second evaluation was carried out five days after withdrawal from cold storage to allow development of chilling injury symptoms as a shelf-life simulation. After the second evaluation fruit were peeled, peel freeze-dried, milled using mortar and pestle and stored at -21°C for further physiological analysis. Freeze-dried peel was analysed for soluble sugars (glucose, fructose, sucrose), vitamin C (ascorbic acid), vitamin E (α-tocopherol), β-carotene, polyamines (putrescine, spermine, spermidine), specific flavanones (naringin and hesperidin) using HPLC-UV-Vis detector and proline, total antioxidant assays (FRAP, ABTS, DPPH), total phenolics, total flavonoids, lipid peroxidation using spectrophotometry, as well as for the heat shock protein (HSP70) using electrophoresis and silver-staining. Chilling susceptibility of lemon fruit varied with fruit source; those sourced from Ukulinga and Eston Estates were chilling resistant, while fruit from Sun Valley Estates showed chilling injury symptoms after 28 days of cold storage plus five days shelf-life. Furthermore, hot water dips (HW) 53°C, 1 μM Molybdenum (Mo) and 10 μM Mo plus HW 53°C significantly reduced chilling injury symptoms compared with the control and HW 47°C. In addition, Sun Valley Estates fruit also showed higher fruit weight loss compared with non-chilling resistant lemons. The alignment of higher fruit weight loss during storage with chilling susceptibility ascertains the use of weight loss as a non-destructive parameter for chilling susceptibility. With respect to flavedo sugars, glucose was found to be the dominant soluble sugar with multi-functional roles during cold storage. This plays a significant role in mitigating cellular stress. Chilling susceptible lemons from Sun Valley Estates had low flavedo glucose concentrations and, therefore, little conversion of glucose to ascorbic acid was possible resulting in a low antioxidant capacity. However, treatments with HW 53°C and Mo soaks seemed to enhance the enzymatic conversion of glucose to ascorbic acid leading to a higher antioxidant capacity in the flavedo of such treated fruit. Furthermore, glucose also feeds into the pentose phosphate pathway which is coupled with the shikimate pathway synthesizing secondary metabolites, especially of the phenolics group. The decrease in glucose was aligned to the levels of total phenolics, but not to that of β-carotene, naringin and hesperidin through 28 days into cold storage period. Moreover, as glucose also feeds into shikimate pathway, simultaneously an increase in proline flavedo concentration was observed. Proline is an antioxidant synthesized from glutamate; as cellular glucose decreases so does the total antioxidant capacity during cold storage. Ascorbic acid is a dominant and potent antioxidant in lemon flavedo as proven with the FRAP, ABTS and DPPH assays. Chilling resistant fruit have significantly higher ascorbic acid conversion. Furthermore, ascorbic acid also acts to generate the α-tocopheroxy radical to further important membrane-bound antioxidant, vitamin E (α-tocopherol equivalent). Furthermore, the DPPH assay was found to be effective in quantifying total antioxidants in lemon flavedo since it detects both lipophilic and hydrophilic antioxidants compared with the ABTS and FRAP assays which are bias to the estimation of liphophilic or hydrophilic antioxidants, respectively. The hot water and molybdenum treatments increased total antioxidants (DPPH assay) with reduced lipid peroxidation 7 days into cold storage and therefore, reduced chilling symptoms in fruit from Sun Valley Estates. The capacity of antioxidant to scavenge reactive oxygen species (ROS) was increased during cold storage and membrane stability significantly improved. Furthermore, putrescine as low valency polyamine was reduced as such compound acted as precursor to the synthesis of the high valency polyamines, spermine and spermidine. Chilling susceptible lemons from Sun Valley Estates showed increased soluble-conjugated polyamines as a response to stress. Furthermore, HW 53°C, 1 μM Mo and 10 μM Mo plus HW 53°C significantly increased the protein concentration and, therefore, likely also the occurrence of proteins with 70kDa (as estimator of HSP70). Additionally, the concentration of conjugated high valency polyamines was also increased, resulting in reduced chilling injury symptoms. The effect of ROS has only been viewed as damaging, while recently their role has also been viewed as stress acclamatory signalling compounds when produced concentrations below critical damaging threshold. Therefore, hot water dips seems to signals synthesis of total protein which include HSPs which then act throughout cold stress to protect other protein and channel other damaged proteins towards proteolysis. While molybdenum increased ROS production below damaging critical threshold, with ROS signalling stress acclimation by further signalling production of bioactive compound with antioxidant properties.