Advances in Applied Agricultural Sciences 2 (2014); 06: 20-29
Chilling injury induces lipid peroxidation and alters the hydrogen peroxide content inpeel and pulp of “Valencia” orange fruit under low temperature storage conditions
Soheila Mohammadrezakhani *1 and Zahra Pakkish 2
1 Master Science (MSc.) Student of Horticultural Science, Shahid Bahonar University, Kerman, Islamic Republic of Iran. 2 Assistant professor, Horticultural Research Institute, Shahid Bahonar University of Kerman, Islamic Republic of Iran.
The “Valencia” orange (Citrus sinensis L.) is the major Citrus crop in Iran. Limiting factors for longer consumption of “Valencia” orange are low storage performance and the appearance of chilling injuries during storage. Previous studies indicated that putrescine and methyl jasmonate treatment could be induced in cold tolerance of harvested orange fruits during storage. This experiment was carried out to determine effects of putrescine and methyl jasmonate on lipid peroxidation and alters the peroxide hydrogen of peel and pulp of “Valencia” orange fruit. The experimental design was a factorial randomized complete-block with three replications. Orange fruits were treated with 0 (control), 2.5 and 5 mM putrescine and 0 (control), 10 and 20 µM methyl jasmonate and then stored at 5±1 °C, 85-90 % relative humidity for 4 months. The results showed, oranges treated with 5 mM putrescine and 10 µM methyl jasmonate were significantly, lowest amount of lipid peroxidation and peroxide hydrogen of peel and pulp and lower chilling injury percentage than non-treated fruits. So, fruits treated with 5 mlM putrescine and combined with 10 µmol methyl jasmonate showed the best effect.
Sweet orange is widely cultivated throughout the world due to its taste and nutritionary value. Chilling injury (CI) is a serious problem during storage of subtropical fruits at low temperatures such as 2 -5°C (Ritenour et al. 2004). In Citrus, as with many other horticultural commodities, chilling sensitivity imposes a major limitation on the postharvest handling of the fruits, since, for some cultivars, such as grapefruit, the storage at relatively high temperatures of 11–13°C is necessary (Kader and Arpaia 2002). Lipids are the major class of biomolecules targeted by reactive oxygen species (ROS) in a membrane. Lipid peroxidation is of concern because it affects the integrity of membrane structure and alters its functions, leading to cell death. The main lipids targeted by ROS are the polyunsaturated fatty acids (PUFA) (Esterbauer et al. 1991). PUFA constitute approximately 50–90 % of the membrane lipids (Douce et al. 1973). One of the biochemical changes occurring when plants are subjected to low temperature stress is the production of ROS which a disruption of the normal metabolism associated with the oxidative damage of lipids, proteins, and other macro molecule (Allen 1995).
A variety of environmental stressors such as soil salinity, drought, extremes of temperature and heavy metals are known to cause oxidative damage to plants either directly or indirectly by triggering an increased level of production of ROS (Malecka et al. 2001). ROS include superoxide radicals (O2-), hydroxyl radicals (OH–) and hydrogen peroxides (H2O2) that are produced as by products during membrane linked electron transport activities as well as by a number of metabolic pathways (Shah et al. 2001). ROS can therefore cause damage to the biomolecules such as membrane lipids, proteins, chloroplast pigments, enzymes, nucleic acids, etc (Mishra and Singhal 1992). Lipid peroxidation is a biochemical marker used for the estimation of free radical mediated injury. ROS–induced stress is thought to be a fundamental cause of cell death in cryopreserved samples (Benson 1990). Superoxide is known to spontaneously produce hydrogen peroxide, a precursor for the formation of the highly reactive hydroxyl radicals (Halliwell and Gutteridge 1999).
Environmental stressor are known to induce hydrogen peroxide and other toxic oxygen species production in cellular compartments and result in acceleration of leaf senescence through lipid peroxidation and other oxidative damages. Hydrogen peroxide as a strong oxidant can initiate localized oxidative damage in leaf cells leading to disruption of metabolic function and loss of cellular integrity that results in senescence. It also changes the redox status of surrounding cells where it initiates an antioxidative response by acting as a signal of oxidative stress (Sairam and Srivastava 2000). Many substances are known to play an important role in protecting plants from free radicals. These are mannitol, sodium formate, sodium benzoate, etc. which are synthesized in various plant parts, and protect plant cells (Shen et al. 1997).
The objective of this study was to determine how putrescine (Put) and methyl jasmonate (MJ) decrease lipid peroxidation and hydrogen peroxide levels and improve chilling tolerance in peel and pulp of orange “Valencia” fruits.
Materials and Methods
Orange fruits (750 fruit) (Citrus sinensis L. var. Valencia) were harvested at full maturity from a commercial orchard at Jiroft (Kerman Province, Iran). Pathogen and mechanical injury free fruits with uniform size and colour were selected, for the experiments. Fruits were treated with 0 (control), 2.5 and 5 mM putrescine and 0 (control), 10 and 20 µM methyl jasmonate and then stored at the temperature of 5±1 °C, with a relative humidity of 85-90 % for 4 months. Chilling injury was visually evaluated by a surface area scale by estimating the percentage of surface pitting and dark patches (browning) according to the following scale: 0 (no signs of surface pitting and dark patches), 1 (<25%), 2 (25-50%), 3 (51-75%) and 4 (>75%). Chilling injury (between 0 and 4) was calculated as [Σ (Chilling injury scale× (number of fruit at the chilling injury level)/ (Total number of fruit in the treatment).
Lipid peroxidation was estimated by determining the malondialdehyde (MDA) content according to the method of Rajinder et al (1981). 100 mg of fruit samples was homogenized in 5 mL of 0.1 % trichloroacetic acid (TCA). The
Table 1: The effect of putrescine and methyl jasmonate on peel hydrogen peroxide values of orange fruit.
hydrogen peroxide value in peel
Methyl jasminate (µM)
*Distinct letters in the row indicate significant differences according to Duncan’s test (P ≤ 0.05).
Figure 1: The effect of Put and MJ on CI of orange fruit. P0J0: Control, P1: 2.5 mM Put, P2: 5 mM Put, J1: 10 µM MJ, J2: 20 µM MJ, P1J1: 2.5 mM Put and 10 µM MJ, P1J2: 2.5 mM Put and 20 µM MJ, P2J1: 5 mM Put and 10 µM MJ, P2J2: 5 mM Put and 20 µM MJ.
homogenate was centrifuged at 10000 g for 5 min at 4 °C. Aliquot of 0.3 mL supernatant was mixed with 1.2 mL of 0.5 % thiobarbituric acid (TBA) prepared in TCA 20 %, and incubated at 95 °C for 30 min. After stopping the reaction in an ice bath for 5 min, samples were centrifuged at 10000 g for 10 min at 25 °C. Absorbance was measured at 532 nm using a Beckman UV-DU 520 spectrophotometer (USA). After subtracting the non-specific absorbance at 600 nm, MDA concentration was determined using the extinction coefficient 155 mM-1 cm-1.
The assay for hydrogen peroxide content was carried out according to Patterson et al (1984), Fresh tissues (2 g) were homogenized with 10 mL of acetone at 0 °C. After centrifugation for 15 min at 6000 g at 4 °C, the supernatant phase was collected. The supernatant (1 mL) was mixed with 0.1 mL of 5 % titanium sulphate and 0.2 mL ammonia, and then centrifuged for 10 min at 6000 g and 4 °C. The pellets were dissolved in 3 mL of 10 % (v/v) H2SO4 and centrifuged for 10 min at 5000 g. Absorbance of the supernatant phase was measured at 410 nm. Hydrogen peroxide content was calculated using hydrogen peroxide as a standard and then expressed as µmol/g-1 on fresh weight basis.
The experimental design was a factorial randomized complete-block with three replications. Data were analyzed by analysis of variance (ANOVA) and the means were compared (p≤0.05) by the Duncan’s multiple range test (DMRT). All analyses were performed by using SAS version 9.1.
Figure 2: The effect of putrescine and methyl jasmonate on pulp hydrogen peroxide of orange fruit. P0J0: Control, P1: 2.5 mM Put, P2: 5 mM Put, J1: 10 µM MJ, J2: 20 µM MJ, P1J1: 2.5 mM Put and 10 µM MJ, P1J2: 2.5 mM Put and 20µM MJ, P2J1: 5 mM Put and 10 µM MJ, P2J2: 5 mM Put and 20 µM MJ.
Figure 3: Correlation between chilling injury and hydrogen peroxide of the orange fruit during storage under low temperature
The percentage of CI on orange fruits as a function of low temperatures for different months are shown in Fig 1. The percentage of injury increased until the 4th months of the experimental period both for treated and non-treated fruits. However the percentage of chilling fruits was lower in treated fruits with Put and MJ compared to control. Therefore, CI symptoms increased during storage both in treated and control fruits, but the CI symptoms in the peel of control fruits was significantly increased compared to treated fruits. There were a significant reduction in browning in fruits treated by 5 mM Put and 10 µM MJ (Fig 1). The amount of hydrogen peroxide of peel and pulp as a function of low temperature for different months are shown in Fig 2 and Table 1. The hydrogen peroxide values increased until the 4th months of the experiment in both treated and non-treated fruits. Hydrogen peroxide content of fruits was lower in fruits treated with Put and MJ than controls (Fig 2 and Table 1). There was a significant decrease in hydrogen peroxide content in fruits treated with 5mM Put and 10µM MJ. Therefore, it seems that hydrogen peroxide content increases as a result of the increase in CI during storage (Fig 3). The effects of Put and MJ on lipid peroxidation content in peel and pulp of oranges subjected to low temperature were given in Fig 4 and 5. In general, storage of orange fruits in low temperatures for 4th months caused a linear increase in lipid peroxidation content. Lipid peroxidation content increased linearly and reached to the highest level in the 4th month of storage in low temperature in controls compared to fruits treated with 5mM Put and 10µM MJ, treatments. Therefore, by increasing chilling stress symptoms during storage, lipid peroxidation content was also increased (Fig 6). However in the present study, exogenous pre-treatment with Put and MJ significantly decreased the lipid peroxidation and hydrogen peroxide content compared to untreated fruits, although an increase in hydrogen peroxide content in orange fruit, was correlated with an in lipid peroxidation content (Fig 7).
The results of the present study showed that oranges treated with 5mM Put and 10 µM MJ were significantly, had significantly lower amount of chilling injury, lipid peroxidation and hydrogen peroxide levels in peel and pulp compared to non- treated fruits. Cell membrane stability has been widely used to express stress tolerance, and higher membrane stability could be correlated with abiotic stress tolerance such as chilling stress (Premachandra et al. 1992). Uemura et al (2006) indicated the necessity of an increase in membrane stability during cold-acclimation both under natural and artificial conditions.
Figure 4: The effect of Put and MJ on peel lipid peroxidation of orange fruit. P0J0: Control, P1: 2.5 mM Put, P2: 5 mM Put, J1: 10 µM MJ, J2: 20 µM MJ, P1J1: 2.5 mM Put and 10 µM MJ, P1J2: 2.5 mM Put and 20µM MJ, P2J1: 5 mM Put and 10 µM MJ, P2J2: 5 mM Put and 20 µM MJ.
Figure 5: The effect of Put and MJ on pulp lipid peroxidation of orange fruit. P0J0: Control, P1: 2.5 mM Put, P2: 5 mM Put, J1: 10 µM MJ, J2: 20 µM MJ, P1J1: 2.5 mM Put and 10 µM MJ, P1J2: 2.5 mM Put and 20µM MJ, P2J1: 5 mM Put and 10 µM MJ, P2J2: 5 mM Put and 20 µM MJ.
Moreover, it was stated that there are compositional, structural and functional changes occurring in the plasma membrane, which result in an increased stability of the plasma membrane under cold conditions. It is known that many of the changes during acclimation to temperature stress are reversible, but if the stress is too great, irreversible changes can occur and these can lead to death (Lester 1985). According to Shilpi and Narendra (2005), symptoms of stress induced injury in plants appear from 48 to 72 h later. However, this duration varies and depends upon the sensitivity of individual plant to cold-stress. The peroxidation reactions differ among the fatty acids depending on the number and position of the double bounds on the acyl chain. Oxidation of unsaturated fatty acids by singlet oxygen produces distinctly different products such as MDA (Bradley and Min 1992). MDA is a common product of lipid peroxidation and a sensitive diagnostic index of oxidative injury (Janero 1990). In this respect increase in lipid peroxidation was reported in many plants under various environmental stresses (Prassad 1996). In the present study, time course of MDA content as an expression of lipid peroxidation was increased by low temperature treatment. However, polyamines (PAs) are involved in many plant physiological processes. It has also been indicated that polyamine protects plant cells against oxidative stress by reducing ROS accumulation (Wang 1994) a finding that is also shown in the present study .In recent research, PA (Put) and MJ have been applied to reduce the development of CI symptoms in orange ‘Valencia’. Thus, Put has a potential application in postharvest treatment by alleviating CI and maintaining quality, decreased lipid peroxidation and hydrogen peroxide production (Serrano et al. 2003). Recently, researchers found that PAs and MJ were effective in alleviating chilling injury of cold stored horticultural crops and this is in accordance with our results. However, the mode of action of PAs and JA in reducing CI and quality deterioration have not been clearly elucidated (Wang 1994). In many studies, it was found PAs (Put) and MJ could counteract oxidative damage and had protective effect against various stressful conditions. According to these experimental results, the exogenous application of these substances have been shown to result in an improved chilling tolerance and reduced incidence of CI in several fruit. In the present study, we found that treatment with them was effective in alleviating CI, lipid peroxidation and hydrogen peroxide production in orange fruit. This finding indicates that the chilling stress tolerance in orange fruit could also be enhanced by postharvest treatment of fruits with Put and MJ. Lipid peroxidation and protective enzyme systems are often evaluated in studies of plant mechanisms under various stressing conditions (van Huystee 1987). Low temperature disrupts the balance of active oxygen species metabolism, such as hydrogen peroxide, leading to their accumulation and destruction of scavenging enzymes such as peroxidases. Alternatively, the accumulation of hydrogen peroxide would also induce lipid peroxidation, damage membrane structure, and cause solute leaking and accumulation of lipid peroxidation (Lee et al. 2001). It has been reported that the improvement of chilling tolerance in harvested horticultural crops is related to enhancement in activities of antioxidant enzymes. The present study also showed that treatment with Put and MJ significantly reduced these CI symptoms, lipid peroxidation and hydrogen peroxide synthesis under chilling stress in orange fruits. Orange fruit is relatively sensitive to CI i.e., they show some symptoms at temperatures 2-5 °C and at these temperatures the symptoms are serious, but storage temperature for oranges are 2-7 °C (Marcilla et al. 2006). The CI symptoms in oranges are usually apparent first in peel than pulp. Stored fruits at low temperature are accompanied by the disruption of surface cells and injury stress of underlying tissues (Hung et al. 2007). Lipid peroxidation and hydrogen peroxide increase permeability that results tissue disruption to and mixing of enzymes and substrates, that affect fruit flavor and texture and reduce shelf life and economic value (Lee et al. 2001). If CI induces membrane damage of organelles such as vacuoles, accumulation of hydrogen peroxide would also induce lipid peroxidation, damage membrane structure, and cause solute leaking and accumulation of lipid peroxidation. In conclusion, our results indicated that Put and MJ could improve the chilling resistance of orange by improving membrane structure and decreasing the accumulation of ROS to protect membranes from chilling damage. Further work is required to clarify whether PAs and JA works by signaling relative genes expression, by acting directly on cell membrane, or by being involved in other unknown processes. Several tropical and subtropical fruit develop CI when exposed to low temperatures.
Figure 6: Correlation between chilling injury and lipid peroxidation of the orange fruit during storage under low temperature
Figure 7: Correlation between hydrogen Peroxide and lipid peroxidation of the orange fruit during storage under low temperature
Our data indicated a correlation between CI, lipid peroxidation and hydrogen peroxide production under low temperature conditions. The considerable increase of CI could lead to an increase of MDA and hydrogen peroxide content, that have deleterious effects under low temperatures, but Put and MJ treatment appear to reduce the percentage of injury. In general levels of MDA and hydrogen peroxide content are significantly increased under low temperatures, which is another indication of cellular damage and CI in orange fruits. However, our results indicated that Put and MJ could improve the chilling resistance of orange by improving cell membrane and decreasing the accumulation of MDA and hydrogen peroxide content to protect membranes from chilling damage. However further work is required to clarify whether PAs and JA works by signaling relative genes expression, by acting directly on cell membrane, or by being involved in other unknown processes.
The authors thank to Faramarz Amiri and Hadi Asghari for supplying the orange fruits used in the study. References
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