BIOCHEMICAL CHARACTERISTIC OF FLORETS SENESCENCE IN BROCCOLI CULTIVARS DURING STORAGE AT LOW AND HIGH TEMPERATURES

Zahra Balouchi¹, Mahmoud Ghasemnezhad¹, Mohammad Saadatian², Gholam Ali Peyvast^1
1 Department of Horticultural Sciences, Faculty of Agriculture, University of Guilan, Rasht, Iran
² Department of General Science, Faculty of Education, Soran University, Soran, Kurdistan Regional Government, Iraq

ABSTRACT

The relationship between oxidative stress and florets senescence of five broccoli cultivars was investigated during two different storage temperatures. Florets were stored three days at 20°C or 40 days at 0°C, followed by two additional days at 20°C. The florets deterioration rate was strongly affected by storage temperature. Subsequently, a rapid decrease of chlorophyll was observed at 20°C. Postharvest senescence of broccoli is correlated with increasing lipid peroxidation (MDA) level, lipoxygenase (LOX) activity and a decline in protein content that has been used as a direct indicator of membrane injury. The antioxidant protection incurred by superoxide dismutase (SOD) and peroxidase (POD) enzymes is important for the retention of green colour in broccoli flower buds and the increases in POD were likely related to florets yellowing. The results showed that storage of the most broccoli cultivars at room temperature (20°C) caused a significant increase in SOD activity, while at 0°C activity of the enzyme declined at the end of 40 days, and thereafter increased at room temperature. The lowest POD and increase in SOD activity in ‘General’ and ‘Revolution’ cultivars at 0°C storage, and ‘Liberty’ and ‘Revolution’ at 20°C, is important for the retention of green colour in florets.

Key words: Brassica oleracea var. italica, senescence antioxidant enzymes, lipid peroxidation, chlorophyll, protein.

INTRODUCTION

Broccoli (Brassica oleracea L. var italica) is a floral vegetable with an important nutritional value due to its content of vitamins, antioxidants and anti-carcinogenic compounds [17, 18, 19]. The inflorescences are harvested, while the floral heads are immature, with the sepals completely surrounding the flower [6, 18]. Therefore, the harvesting induces a severe tissue stress that triggers the senescence process [18]. The most obvious feature of broccoli postharvest senescence is sepal degreening due to chlorophyll degradation [25]. Fresh broccoli has a shelf life of 3–4 weeks in 0°C and 95% RH, but only 2–3 days at 20°C [15]. This is due to broccoli having a relatively high rate of metabolism and consequently a high respiration rate, being extremely sensitive to ethylene [15]. Its deterioration rate appears to be affected by storage temperature [31] .

In plants, senescence is often accompanied by morphological changes and alterations in biochemical and biophysical properties of metabolism [14] and degradation of macromolecules such as proteins, nucleic acids and lipids, loss of chlorophyll [22]. Furthermore, it is associated with excess production of reactive oxygen species (ROS), including superoxide radicals and hydrogen peroxide (H₂O₂) [21]. Oxidative stress will occur when the generation of ROS exceeds the antioxidant capacity of the cells [14]. Thompson et al. [26] have shown that lipid peroxidation is mediated via oxygen radicals. According to Zhuang et al. [30], postharvest senescence of broccoli is correlated with lipid peroxidation level, and has been used as a direct indicator of membrane injury [28]. Lipoxygenase (LOX) enzymes are regarded to be responsible for membrane degradation, because they catalysis the peroxidation of polyunsaturated fatty acids [28]. Plant possesses a well-defined enzymatic antioxidant defense system to protect themselves against these deleterious effects by scavenging ROS [2]. Toivonen and Sweeney [27] reported that antioxidant protection offered by Superoxide dismutase (SOD) and Peroxidase (POD) is important for the retention of green color in broccoli flower buds and the increases in SOD and POD were likely responses to the increases in oxygen radical production in broccoli, which could subsequently lead to yellowing. This increase has been found in previous work [9, 23]. Starzyńska et al. [24], reported that storage of broccoli at room temperature caused a significant increase of POD and SOD activity, while at low temperature (5°C) activity of the enzyme rose after 10 days. The rapid decrease of chlorophyll was observed only in the case of broccoli heads stored at 20°C.

The objective of the present study was to investigate some biochemical changes during florets senescence and their relationship during low and room temperature storage.

MATERIALS AND METHODS

Plant material
Five different broccoli (Brassica oleracea var. Italica) cultivars, ‘General’, ‘Liberty’, ‘Pilgrim’, ‘Revolution’ and ‘Millady’ were grown at University of Guilan, Rasht, Iran. The growth condition was adequate and similar for all cultivars. The inflorescences were harvested in the early morning, when the heads were completely developed and without opened florets [11]. Head was separated into florets and stem and disinfected with chlorinate water (150 ml/l as sodium hypochlorite) for 15 min, followed by repeated washing with distilled water. After drying at room temperature, Broccoli florets were dried at room temperature, then three florets of broccoli (include 20 g each florets) were placed in polyethylene bags with overall dimensions 20×20 cm² and 29.2 pmol/s/m²/Pa oxygen transmission rate film and stored three days at 20°C and at 0°C for 40 days, followed by two additional days at 20°C. The following biochemical characteristics were evaluated at harvest time (day 0), and 40 days as well as 2 additional days in 0°C storage, and during three days at 20°C.

Laboratory Evaluation
Total chlorophyll content was measured according to Lemoine et al. [20]. Florets were powdered by liquid nitrogen in a refrigerated mill and 0.4 g of the powder obtained was added to 5 ml of acetone/water (80:20), stirred and then centrifuged at 5000 rpm for 15 min. The supernatant was used to determine the chlorophyll content. Results were expressed as mg chlorophyll g /100 g FW.

The protein concentration of the supernatant was estimated using the method of Bradford [5]. The absorbance was read at 595 nm using UV-visible spectrophotometer model PG Instrument +80, England. The amount of protein was quantified by using a standard curve and result were expressed as mg protein per g fresh weight of floret.

Lipid peroxidation (MDA level) was determined by the method of Heath and Packer [13]. Floret sample (0.5 g) was homogenized in 1 ml of 0.1% trichloracetic acid (TCA). The homogenate was centrifuged at 14 000 rpm for 15 min, and then 600 µl of supernatant was added to 600 µl ml of 0.5% thiobarbituric acid (TBA) in 20% TCA. The mixture was heated at 95°C for 30 min and then cooled in an ice bath. After centrifugation at 10 000 rpm for 10 min, the absorbance of the supernatant was recorded at 532 nm. The MDA level was calculated according to its extinction coefficient of 155 mmol/cm.

Enzyme extract of LOX, SOD and POD was prepared by first freezing a weighed amount of floret tissue (0.5 g) in liquid nitrogen followed by grinding with 10 ml extraction buffer (50 mmol phosphate buffer, pH 7 containing 0.5 mmol EDTA and 2% PVPP (w/v). Homogenate was centrifuged for 20 min at 15 000 rpm and the supernatant used to determine enzymes activity.

LOX activity was estimated according to the method of Bonnet and Crouzet [4] with some modification. The substrate solution was prepared by adding 10 µl linoleic acid to 4 ml distilled water containing 5 µl Tween-20. The solution was kept at pH 9.0 by adding 1 ml NaOH 0.2 mol until all the linoleic acid was dissolved and the pH remained stable, and the total volume was adjusted until 25 ml. LOX activity was determined spectrophotometrically by adding 20 µl of enzyme extract to 80 µl linoleic acid substrate and 900 µl phosphate buffer solution (50 mmol, pH 7). Absorbance was recorded at 234 nm for 10 min at 30°C (extinction coefficient, 25mol/cm).

SOD activity was estimated by recording the decrease in absorbance of nitro-blue tetrazolium dye (NBT) [9]. Three ml of the reaction mixture contained 13 mmol methionine, 25 mmol NBT, 0.1 mmol EDTA, 50 mmol phosphate buffers (pH 7.8), 50 mmol sodium carbonate and 0.1 ml enzyme. The reaction started by adding 2l mol riboflavin and placing the tubes under two 15 W fluorescent lamps for 15 min. The complete reaction mixture without enzyme, which yielded maximal color, acted as the control. The reaction stopped by switching off the light and putting the tubes in the dark. A non-irradiated reaction mixture served as a blank. The activity is expressed as an unite per mg fresh weight. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the rate of NBT photoreduction.

POD activity was assayed by spectrophotometrically measuring the formation of guaiacol in l ml reaction mixture of 450 μl 25 mmol guaiacol, 450 μl 225 mmol H₂O₂ and 100 μl crude enzymes. The increase in absorbance was recorded by the addition of H₂O₂ at 470 nm for 3 min (e, 26.6 mmol /cm).

Statistical Analysis
All determination was performed in triplicate. The recorded data were statistically analyzed (ANOVA analysis) using the software of SAS. Sources of variation were five different hybrid broccoli cultivar and two storage conditions. Means were compared with the Tukey’s test at p ≤ 0.05.

RESULTS

Chlorophyll content
Regardless of broccoli cultivars, chlorophyll content of florets declined during storage (Tab. 1, 2). The rapid decrease of chlorophyll was observed only in the case of broccoli heads stored at 20°C, but low storage temperature delayed yellowing and reduced loss in chlorophyll concentration. The ‘General’ cultivar maintained the higher chlorophyll content after 40 days storage at 0°C and two additional days at 20°C (Tab. 2), but at room temperature (20°C) storage, chlorophyll content of ‘Pilgrim’ was higher than others cultivars (Tab. 1).

Although, chlorophyll content of ‘General’ , ‘Millady’ and ‘Pilgrim’ was higher at harvest time than two other cultivars (Tab. 1, 2), but the decline in total chlorophyll was much greater for these cultivars than ‘Liberty’and ‘Revolution’ at 20°C storage (Tab. 1). In contrast, ‘General’ and ‘Revolution’ retained chlorophyll content over 40 days at 0°C and two additional days at 20°C (Tab. 2). Therefore ‘General’and ‘Revolution’ cultivars at 0°C storage and Liberty’ and ‘Revolution’ at 20°C retained stable chlorophyll content during storage. In both temperature conditions, there was a positive correlation between chlorophyll content with protein and SOD, and a negative correlation with MDA and POD (Tab. 3, 4).

Lipid peroxidation (MDA)
The lipid peroxidation level increased during storage both at room temperature and low temperature storage, followed by two additional days at room temperature in florets broccoli cultivars (Tab. 1, 2). All broccoli cultivars showed a significant increase in MDA level after removal to the room temperature, but there was no significant difference found among the broccoli cultivars. Lipid peroxidation was increased as florets progressively lost chlorophyll (r= 0.57 and r=0.73 for room and low temperature storage respectively (Tab. 3, 4).

Protein content
The changes of protein content in broccoli florets during two different storage conditions were summarized at Table 1 and 2. There was no significant difference between cultivar and storage condition, although a decline in protein content was at the both temperature conditions. The protein content reduction was correlated with chlorophyll loss (r= 0.37 and r=0.53 for room and low temperature storage respectively) and with enhancement lipid peroxidation (r= 0.36 and r=0.42 for room and low temperature storage respectively) (Tab. 3, 4).

Lipoxygenase activity (LOX)
The changes of LOX in broccoli florets during three days storage at 20°C and during 40 days storage at 0°C, followed by two additional days at 20°C are found at figure 1 and 2. During storage, no significant changes were observed in LOX activity. However, LOX activity gradually increased during storage at 20°C at the all broccoli cultivars. The highest LOX activity was found in ‘Millady’ and ‘General’ cultivar during two different storage conditions was observed. Increasing LOX has been associated with enhancement of lipid peroxidation in room temperature (r = 0.35) (Tab. 3).

Superoxide dismutase (SOD)
Storage of the most broccoli cultivars at room temperature (20°C) caused the significant increase of SOD activity, while at low temperature (0°C), activity of SOD declined after 40 days, but increased after removing to room temperature (Fig. 3, 4). The SOD activity in ‘General’ , ‘Millady’ and ‘Revolution’ cultivars decreased after harvest time, but followed by its increase (Fig. 3). Also, the SOD activity in ‘General’ cultivar decreased during storage (Fig. 3).

The SOD activity decreased in ‘General’ , ‘Revolution’ and ‘Millady’ cultivars by 40 days, and thereafter, increased after removal to room temperature (Fig. 4). In ‘Pilgirim’ cultivar with over time SOD activity increased, while in ‘Liberty’ cultivar decreased. Highest SOD activity after removal to room temperature was recorded in the ‘Revolution’ and ‘General’ cultivar (Fig. 4). The reduction SOD activity was associated with enhancement of LOX activity (r = 0.24 and r=0.28 for room and low temperature storage respectively) (Tab. 3, 4). Furthermore, in low temperature storage correlation between SOD activity with chlorophyll content was positive (r=0.28), but with MDA and POD was negative (r=0.36 and r=0.28 respectively).

Peroxidase (POD)
The results showed that storage of broccoli florets both at room temperature (20°C) and low temperature (0°C) caused the significant increase in POD activity (Fig. 5, 6). The rapid increase in POD activity was observed after removing to 20°C. Storage of florets at room temperature caused the increase of POD activity in ‘General’ , ‘Pilgrim’ and ‘Millady’ cultivars (Fig. 5). The highest activity of this enzyme was found in the ‘General’ and ‘Millady’cultivars at the end of the storage (Fig. 5).

In low temperature storage, POD activity increased significantly compared to the initial value in all cultivars exception ‘General and ‘Revolution’ cultivar. No significant differences were recorded in other cultivars at the end of the storage. The POD activity was associated with enhancement of lipid peroxidation (r=0.53 and r=0.51 for room and low temperature storage respectively) and with chlorophyll loss (r=0.43 and r=0.43 for room and low temperature storage respectively) (Tab. 3, 4).

DISCUSSION

Chlorophyll content
Chlorophyll loss has been associated with lipid peroxidation [31] and with enhancement of POD activity [7]. Changes of chlorophyll level in photosynthetic cells are good indicators of senescence, occurring in green vegetables after harvested. Chlorophyll content degradation increased with temperature in broccoli florets [24] and detected pakchoy leaves [1]. These results demonstrated that the temperature has an important role in florets senescence in broccoli [16, 11] and in some instances, influence the rate of senescence as measured by chlorophyll loss [14]. Starzyńska et al. [24] reported that chlorophyll was degraded at a slower rate in the broccoli stored at 0°C, when compared with the material stored at room temperature. Low storage temperature delayed yellowing and reduced loss in chlorophyll concentration [24].

Lipid peroxidation
Malondialdehyde (MDA) is the product of membrane peroxidation and has been used as a direct indicator of membrane injury [28]. The peroxidation of lipids can be initiated usually by free radical [27]. Thompson et al. [26] have shown that lipid peroxidation is mediated via oxygen radicals, particularly hydroxyl radicals. Furthermore, lipid peroxidation leads to the generation of free radicals which in turn initiates an increase in ethylene formation leading to the promotion of senescence [31]. It has been reported that the membrane permeability and the level of MDA content increaseed during tabacum leaves [9] and broccoli [28] senescence. Zhuang et al. [29] have reported that increased temperatures accelerated the deterioration and lipid peroxidation of broccoli buds increased rate generation of free radicals and ethylene. Therefore, an increase in MDA production is usually considered to be an oxidative stress indicator in plants and a result of senescence [14]. The rapid increase of MDA content in flower bud tissue indicates the importance of the influence of storage temperature in induction senescence in broccoli florets tissues.

Protein content
A loss of protein has been reported during senescence in fruits and vegetables [31, 14, 8, 18]. It’s suggested during senescence, loss of integrity and functionality of membranes may lead to solubilization of anchored proteins [18]. The solubilized proteins are utilized as respiratory substrates, leading to a decrease in the protein content [18]. Thus, low temperature could decrease the intensity of the respiration of flower bud cells, and, as a consequence, could slow down the ROS, intensity of metabolic processes, lipid peroxidation, and protein degradation.

Lipoxygenase activity (LOX)
Increased activities of LOX during senescence associated with membrane polyunsaturated fatty acid peroxidation [14, 28] and formation of further free radicals in some plant tissues [14]. LOX catalyses the oxygenation of polyunsaturate fatty acids to form fatty acid hydroperoxides [3, 28]. The rise of LOX activity during storage has also been reported by Zhuang et al. [31] and Yuan et al. [28]. It’s suggested during senescence, loss of integrity and functionality of membranes may lead to solubilization of anchored proteins [18]. The solubilized proteins are utilized as respiratory substrates, leading to a decrease in the protein content [18]. Thus, Low temperature could decrease the respiration rate of flower bud cells, and, as a consequence, could slow down the ROS, intensity of metabolic processes, lipid peroxidation and protein degradation.

Superoxide dismutase (SOD)
SOD activity is regarded as the key enzymatic antioxidant in plant, which increases in stress conditions [24]. The increase of SOD in storage condition associated with the rate of oxygen radical that is produced in stress condition [23]. SOD increase in harvest time was response to the stress due to harvest. With time and reduce the stress caused by the harvest, SOD activity is decreased [11]. The result of higher SOD activity is an intensified synthesis of hydrogen peroxide the signal molecule, which induces activities of other enzymatic antioxidants [24]. The increase of SOD activity in broccoli flower buds after removal to 20°C temperature is likely a response to increases in oxygen radical production in cells, caused by the storage stress. Thus, the increase in SOD activity may be a factor of better plant tolerance to unfavorable environmental [24]. Dhindsa and Thorpe [9], in experiment conducted on tubacum leaves, showed that the increase in this enzyme activity is characteristic for plant tissue senescence. With broccoli, the increase of SOD activity in flower buds taken from heads stored at 13°C for 4 days found also Toivonen and Sweeney [27].

Peroxidase (POD)
POD enzyme plays an important role in the fine regulation of ROS concentration in the cell through activating and deactivating H₂O₂. POD catalyzes the decomposition of H₂O₂ to H2O [14]. The changes in its activity are induced by the substrate level superoxide radical, which increases in stress [24]. Therefore, POD could be involved in the degradation of chlorophyll [10]. Thus, induction of peroxidase activity is a well known indicator of stage of senescence and intense stress [24]. The increments of POD activity during broccoli senescence were previously reported. Funamoto et al. [12] reported that POD activity increased markedly during yellowing of broccoli, the lowest POD activity associated with the highest chlorophyll content and the highest POD activity determined with the lowest chlorophyll content. The increase in POD is likely a response to the increases in oxygen radical production in the broccoli, which could subsequently lead to yellowing [27]. Starzyńska et al. [24] reported that storage of broccoli at room temperature caused the significant increase in POD activity, while at low temperature (5°C) activity of the enzyme rose after 10 days..

CONCLUSION

The results demonstrate the importance of storage temperature and cultivar on biochemical response and the rate of senescence as measured by chlorophyll loss. The rapid decline in chlorophyll content indicates high oxidative damage that in high temperatures is more rapid than in low temperature. The ‘General’ and ‘Revolution’ cultivars with maintenance of stable chlorophyll content and low POD and high SOD activity, respectively, during 40 days storage at 0°C, followed by two additional days at 20°C that was suitable for the long-term storage, while during short-term storage at room temperatures, with the lowest and highest POD and SOD, respectively, was observed in the ‘Liberty’ and ‘Revolution’ cultivars that were associated with maintenance higher chlorophyll content . Correlation between biochemical characteristics indicate that POD can be an important factor in the accumulation of MDA and protein degradation, and causes the yellowing of broccoli. Finally, low temperature could decrease the rate of biochemical processes in flower buds, and thus could slow generation of ROS, diminish lipid peroxidation and protein degradation.

Acknowledgements

The authors are grateful to the University of Guilan in Iran for financial support.

REFERENCES

Accepted for print: 15.06.2014

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