Postharvest Application of 1-Methylcyclopropene Maintains Quality and Extends the Shelf-Life of Zizania latifolia during Storage at 25˚C ()
1. Introduction
Zizania latifolia, belongs to Oryzae family, has been harvested for more than thousands of years and used as an aquatic crop [1] . After, infection by the fungus Ustilago esculenta, the young shoots appear edible, soft and swollen. It also has a large nutritional and economical importance [2] [3] . Two varieties of Z. latifolia are cultivated in China namely: the one harvested once a year (single season culture) and the two seasons culture harvested twice a year in summer and autumn [1] [4] .
The natural environment of Z. latifolia has shifted with the growth of human population and the drying of rice lakes, which have both limited the space covered by these species [5] . The largest area under harvesting in China is mostly situated in the area surrounding Taihu Lakes, in Jiangsu and Zhejiang provinces [1] . In addition, Yan et al. [5] stated that Z. latifolia, is prevalent throughout China except in Xinjiang and Tibet, and grows in lakes marshes, ponds, rivers and the wetlands, particularly abundant in Huai river basin and the middle and lower Yangtze river basins.
Z. latifolia is an important vegetable with high nutritional and economical values and also used to prevent and treat metabolic disease [5] . However, abiotic and biotic stresses can reduce its shelf-life during postharvest storage. In addition, tissue lignification, cut surface browning, and yellowing are considered as main causes of its quality loss [6] [7] [8] [9] . Therefore, effective postharvest treatments that can delay senescence and control the quality deterioration are needed. Previous study was based on the use of edible coating [10] , high pressure CO2 [11] for the storage of fresh-cut Z. latifolia and cold storage at 1˚C of whole Z. latifolia as well as the ultraviolet-C [12] , nitric oxide [13] [14] , melatonin [15] and ethanol vapor [16] treatments. However, little information regarding the use of 1-MCP on the physiological and cell ultrastructure changes of postharvest storage of Z. latifolia was available.
1-Methylcyclopropene (1-MCP), as an inhibitor of ethylene action, has been shown to passivate receptors by competing with ethylene to inhibit ethylene production and release, thereby preventing physiological and biochemical activities related to maturation [17] . 1-MCP provides commercial potential for controlling ripening, aging, yellowing, and softening caused by ethylene, and extends the shelf life of some vegetables, including pak choi, green pepper, bitter gourd, sweet basil leaves, and okra [18] [19] [20] [21] . Song et al. [18] applied 1-MCP treatment after harvesting Brassica rapa subsp. chinensis and found that the treatment could delay the yellowing of the leaves of Brassica rapa subsp. chinensis by maintaining the integrity of the chloroplast structure during storage at 20˚C. Hassan et al. [20] fumigated young immature bitter melons with different concentrations of 1-MCP for 12 hours and proved that 5 µl∙L−1 1-MCP treatment significantly improved the fruit quality of bitter melons and maintained the activity of antioxidant enzymes in bitter melons during early storage. Besides, the beneficial effects of 1-MCP on plant aging also include inhibition of antioxidant enzyme activity [17] and control of ROS production [22] .
The purpose of this study was to investigate the effect of 1-MCP treatment on the reactive oxygen metabolism and ultrastructure of Z. latifolia during postharvest storage at room temperature (25˚C). The study specifically based on the effect of 1-MCP on changes in overall appearance quality, physiological and biochemical parameters such as antioxidant system and lipid metabolism. The results can help to better understand the mechanism of 1-MCP treatment on the regulation of Z. latifolia postharvest senescence and provide a technical support for maintaining the quality and extending the shelf-life of Z. latifolia.
2. Materials and Methods
2.1. Plant Material
Fresh Z. latifolia stems were hand-harvested from a commercial farmland in Yixing, Jiangsu, China. They were selected on the basis of similarity in size, color and absence of visible defects and then arbitrarily divided into two groups of 15 kg vegetables each. The first group of vegetable was treated with 10 µl∙L−1 1-MCP (Agrofresh, USA) in a sealed chamber at room temperature (25˚C) for 20 h; the second group (CK) was subject to the same condition without exposure to 1-MCP. Following treatment, the chambers were opened and two groups of vegetable were stored at room temperature for 6 days. Samples were taken out at 0 (before treatment, CK0), 1 (CK1, 1-MCP1), 3 (CK3, 1-MCP3), 6 (CK6, 1-MCP6) day, respectively, and manually peeled carefully to remove boots. Next, about 5 cm stem portion was removed from each end of an individual with a sharp stainless-steel knife and the remainder was used for indexes analysis, or directly frozen in liquid nitrogen and stored at −80˚C until further analysis.
2.2. Color Determination
The Minolta CR-200 portable colorimeter was used to measure Z. latifolia’s CIE L, a*, b* values. Brightness was represented by L value (L = 0, black; L = 100, white), positive a value represented the degree of redness, negative a* value represented the degree of greenness, b*+ represented the degree of yellowness and b represented the degree of blueness.
The total color difference (∆E) was calculated as follows:
according to the method of Gouda et al. [23] (where, L0,
and
are the readings at the beginning of storage, and L, a* and b* are the individual readings at each storage time point thereafter).
2.3. Weight Loss and Respiration Rate
The weight loss assessment was made using the following formula: Weight loss rate% = (weight before storage − weight after storage) × 100/weight before storage.
The CheckMate 3 portable headspace analyser (PBI Dansensor, Denmark) was used to determine the percentage of O2 consumed and the volume of CO2 generated per unit time in a fixed-volume airtight container. The result was expressed as mg CO2∙Kg−1∙h−1.
2.4. Visualization of Ultrastructure
The visualization Z. latifolia cell ultrastructure was conducted using the method of Li et al. [24] with some modification. Z. latifolia pieces were collected from the cut surface of three Z. latifolia per treatment. The pieces were resuspended in phosphate buffered saline (PBS) containing 5 mM cerium chloride (CeCl3), incubated at 28˚C for 1.5 h after pretreatment. Cells were collected by centrifugation and the supernatant containing residual CeCl3 was discarded. The cells were resuspended in 3% glutaraldehyde for more than 4 hours, and the cells were pelleted by centrifugation. After fixing with OsO4 and embedding with epoxy resin, the slices were sliced, and the slices were observed under 80 kV with a transmission electron microscope.
2.5. Ascorbic Acid (AsA) Content Determination
The AsA content was measured using the 2,6-dichlorophenol indophenol method as stated by Li et al. [25] . The result was expressed as gram of AsA per kilogram on a fresh weight basis.
2.6. Determination of Superoxide Anion (
), Hydrogen Peroxide (H2O2) and Malondialdehyde (MDA)
The
production rate, H2O2 and MDA contents were determined using the method of Gouda et al. [23] . The results were expressed as μM∙kg−1∙min−1, mM∙kg−1 and μM∙kg−1, respectively.
2.7. Determination of Superoxide Dismutase (SOD), Catalase (CAT) and Ascorbate Peroxidase (APX)
The SOD, CAT and APX activities were evaluated following the method reported by Luo et al. [6] . One unit of SOD activity was defined as the quantity of enzyme that inhibited 50% of the photo-reduction of NBT at 560 nm. The CAT and APX activity were expressed as the change in A240 and A290 per minute per gram of fresh weight, respectively.
2.8. Determination of Phenylalanine Ammonia Lyase (PAL) and Peroxidase (POD)
The standard procedure reported by Gao et al. [26] was used for the determination of PAL and POD activities. One unit of PAL and POD activities were defined as the amount of enzyme that caused an increase in absorbance of 0.01 at 290 nm in 1 h, and at 460 nm in 1 min, respectively.
2.9. Determination of Phospholipase D (PLD), Lipoxygenase (LOX) and Lipase
The method used to determine the enzymatic activity of PLD was the usual protocol documented by Song et al. [18] . A 0.01 change in the absorbance (A520, A243, A520,) value per minute were defined as a unit of PLD, LOX and lipase enzyme activity, respectively.
2.10. Statistical Analysis
Physiological data were performed as mean ± standard deviation of three replicate samples. SPSS 22.0 software and the Duncan’s multiple range test were used to perform one-way analysis of variance and to determine differences in each treatment at p = 0.05. The process was repeated three times for each treatment, and the results were expressed as the mean ± standard deviation.
3. Results
3.1. Effect of 1-MCP on Visual Appearance Quality of Z. latifolia during Storage
As indicated in Figure 1, the control sample of Z. latifolia presented a better appearance with a bright green shell, white skin and internal color at day 0, but after 6 days of storage at room temperature, the shell turned yellow and its epidermis was browned and blackened. In addition, some hollow bran appeared and there were signs of deterioration. However, after 6 days of storage, sample treated with 1-MCP retained an excellent color and visual appearance.
3.2. Effect of 1-MCP on Color Change during Storage at 25˚C
As seen in Table 1, there is no significant difference of L value between the treated and control sample, however, the treated sample kept lower the a*, b* and ∆E values throughout the storage time comparing with those of the control sample.
3.3. Effect of 1-MCP on the Z. latifolia Cell Ultrastructure
As observed in Figure 2, the cell structure of Z. latifolia is intact at day 0 with
Figure 1. Effect of 1-MCP on visual appearance quality of Z. latifolia during storage at 25˚C ((A) CK 0 d; (B) CK 1 d; (C) CK 3 d; (D) CK 6 d; (E) 1-MCP 1 d; (F) 1-MCP 3 d; (G) 1-MCP 6 d).
Table 1. Effect of 1-MCP on L, a*, b* and ∆E values changes during Z. latifolia storage at 25˚C.
Data are expressed as means ± SD of triplicate assays. Within the same index, mean values with different lowercase letters in same column are significantly different (p < 0.05).
Figure 2. Effect of 1-MCP treatment on cell ultrastructure of Z. latifolia during storage at 25˚C ((A) CK 0 d; (B) CK 1 d; (C) CK 3 d; (D) CK 6 d; (E) 1-MCP 1 d; (F) 1-MCP 3 d; (G) 1-MCP 6 d).
visible cell wall, nucleus and mitochondria. However, after 6 days of storage, the cell wall and plasma membrane of the control sample was significantly degraded with unclear mitochondria. Meanwhile, the treated samples kept their cell wall and plasma membrane intact with a visible and abundant mitochondria. These observations indicated that 1-MCP treatment has positive effect in the maintenance of Z. latifolia cell ultrastructure.
3.4. Effect of 1-MCP on the Weight Loss and Respiratory Intensity of Z. latifolia during Storage
Weight loss in vegetable and fruit is related to the loss of water caused by transpiration and loss of carbon due to the respiration processes. Therefore, in our study, the weight loss and respiration rate were monitored (Table 2).
As seen in Table 2, throughout the storage time, the weight loss in sample treated with 1-MCP was lower than that in control sample. This indicated that 1-MCP treatment had a better performance on inhibition of water loss. However different pattern was observed concerning the respiratory rate. 1-MCP treatment maintained lower the respiratory rate of Z. latifolia during the first three days. This decrease trend was followed by an increase of respiratory rate which led to high respiratory rate of 1-MCP treated sample than that of the control group (Table 2).
3.5. Effect of 1-MCP on
, H2O2 and MDA Contents
According to Table 2, there is an increase and decrease trend of
production rate and H2O2 accumulation in both control and treated samples. Interestingly, sample treated with 1-MCP shown the lower content of H2O2 and
through the storage period comparing to the control. In addition, the Table 2 showing the content of MDA during storage indicated the lower MDA content in samples treated with comparing with the control group along with the storage time prolongs. These data suggest that 1-MCP treated had a positive impact on inhibiting
production, H2O2 and MDA accumulation in Z. latifolia during storage at 25˚C.
3.6. Effect of 1-MCP on SOD, CAT and APX Activities and AsA Content
Table 3 shown that APX activity of Z. latifolia decreased in the early period of
Table 2. Effect of 1-MCP on the weight loss, respiratory intensity,
production rate, H2O2 and MDA contents of Z. latifolia during storage at 25˚C.
Data are expressed as means ± SD of triplicate assays. Within the same index, mean values with different lowercase letters in same column are significantly different (p < 0.05).
storage and then increased in both control and treated samples. However, the APX activity of samples treated with 1-MCP (48.00 ± 1.05 U∙g−1) was higher than the control samples (43.60 ± 1.67 U∙g−1). In addition, samples treated with 1-MCP shown the highest SOD and CAT activities (Table 3) throughout the storage compared with the control samples. As indicated in Table 3, there is a decrease of AsA content in the control and treated sample during the storage time. However, the AsA content in the treated samples was still higher than that of the control group at the end of storage period. These results suggested that 1-MCP treatment was efficient to maintain higher the activity of antioxidant enzymes.
3.7. Effect of 1-MCP on POD, PAL Activities
As seen in Table 4, POD activity significantly increased in the control samples throughout storage while the POD activity in the treated samples decreased at day 1 and 3 and increased up to 573.1 ± 66.04 U∙g−1 at day 6, which is lower than that on the control samples (581.5 ± 20.85 U∙g−1). The same pattern was observed in Table 4 where sample treated with 1-MCP presented a lower PAL enzyme activity during the whole storage period.
3.8. Effect of 1-MCP on LOX, PLD and Lipase Activities
In order to gain a better understanding regards the effect of 1-MCP during the postharvest storage of Z. latifolia, three membranes lipids degrading enzymes including PLD, lipase and LOX activities were investigated and represented. Lipase has an effect on the membrane lipids of fruits and vegetables. PLD affects the structure, function and stability of the membrane by hydrolyzing phospholipids in the cell membrane. LOX is closely involved in the lipid action of fruit and
Table 3. Effect of 1-MCP on APX, SOD, CAT activities and AsA content of Z. latifolia during storage at 25˚C.
Data are expressed as means ± SD of triplicate assays. Within the same index, mean values with different lowercase letters in same column are significantly different (p < 0.05).
Table 4. Effect of 1-MCP on POD and PAL activities of Z. latifolia during storage at 25˚C.
Data are expressed as means ± SD of triplicate assays. Within the same index, mean values with different lowercase letters in same column are significantly different (p < 0.05).
Table 5. Effect of 1-MCP on lipase, PLD and LOX activities of Z. latifolia during storage at 25˚C.
Data are expressed as means ± SD of triplicate assays. Within the same index, mean values with different lowercase letters in same column are significantly different (p < 0.05).
vegetable cells. And Lipase, PLD and LOX is considered an important enzyme that causes post ripening and aging of fruits and vegetables [22] . As seen in Table 5, PLD activity significantly increased from 70.14 ± 0.017 U∙g−1 at day 0 to 137.57 ± 0.016 U∙g−1 and 103.86 ± 0.013 U∙g−1 at day 3 in the control samples and treated samples respectively. In addition, the PLD enzyme activity of both groups significantly decreased with the lower value in the treated group (93.71 ± 0.027 U∙g−1) at the end of storage. Meanwhile, during the storage period, decrease of lipase activity was observed in the control and treated sample (Table 5). However, the sample treated with 1-MCP showed the lower lipase activity. Moreover, LOX activity increased during the early storage time in both samples to reach the values of 52.04 ± 1.46 U∙g−1 in the control samples and 40.52 ± 2.29 U∙g−1 in 1-MCP treated samples at the end of storage (Table 5).
Taken together, PLD, lipase and LOX activities in 1-MCP treated group were finally lower than that of the control groups after the storage. These data indicate that 1-MCP treatment significantly inhibited the PLD, lipase, and LOX activities.
4. Discussion
Senescence is known as a series of active degenerative processes of a cell and organism that are under genetic control. The loss of visual appearance of vegetables could be the result of senescence and considered as a negative attribute in commercial condition [27] . 1-MCP treatment has been reported to delay the weight loss and maintain a good visual quality appearance of horticultural products such as yardlong bean [28] , cherry tomato [29] , mango fruit [30] . In addition, 1-MCP treatment had a positive effect on delaying the weight loss during the postharvest storage of sweet basil leaf [20] . In our study, 1-MCP treatment significantly retarded the weight loss and maintained the visual appearance as well as preserved the color of Z. latifolia by keeping lower a, b and ∆E values as well as delaying the formation of hollow bran. These results are consistent with the findings of Luo et al. [31] .
In addition, previous study reported the effect of 1-MCP on the inhibition of respiration rate [22] [32] . It was reported that 1-MCP treatment could reduce the respiration rate of horticultural products during storage. However, our current finding showed that sample treated with 1-MCP have a lower respiration rate during the 3 first day which become higher than the control samples rate after 6 days of storage. Similarly, Mccollum et al. [33] found that grape fruit treated with 75 nL∙L−1 1-MCP had higher respiration rate than those treated with 15 or 30 nL∙L−1 1-MCP. Therefore, they conclude that 1-MCP can retard or increase the respiration rate in grape fruit according on the concentration and time after treatment. In addition, 1-MCP treatment had no significant effect on respiration rate during the storage of “shatangu” mandarin at the end of storage (20 days) at 20˚C [34] .
In our study 1-MCP treatment kept higher the total amount of AsA which is known as a crucial nutritional component in horticultural products with positive biological activity in human body [35] . A high amount of AsA in horticultural products was found to be important on the prevention of brown pigments synthesis. This result was in agreement with the positive effect of 1-MCP retarding the decrease of AsA in yardlong bean [28] .
The senescence of horticultural products during postharvest storage is related to the accumulation of ROS [36] . In addition, previous study reported that an abundance of ROS may induce oxidative reactions with lipids, thereby, inducing the production of harmful substances including MDA. MDA content is commonly considered as indirect indicator of membrane integrity, and a high content may imply loss of membrane integrity [23] . Loss of membrane integrity in fruits and vegetables is an indicator of increased activity of membrane-related lipolytic enzymes, including PLD, lipase and LOX. PLD hydrolyzes phospholipids to phosphatidic acid and diacylglycerol, which are subsequently degraded to free fatty acids by lipase whereas LOX catalyzes the peroxidation of polyunsaturated fatty acids [35] [37] . A positive correlation between LOX activity, MDA content and ROS level was observed by Chomkitichai et al. [38] during the storage of “Daw” longan fruit. In our current study, it was seen that the contents of ROS (
, H2O2) were significantly inhibit by 1-MCP which therefore led to the lower levels of MDA content, PLD, lipase and LOX activities. Similar effect of 1-MCP on the inhibition of ROS was also observed in baby squash treated with 1-MCP [39] . Additionally, Huang et al. [21] found that 1-MCP treatment significantly delayed the loss of membrane integrity in Okra (Hibiscus esculentus) during storage at 7˚C for 18 days. Our findings are also consistent with those of Wang et al. [37] .
At the same time 1-MCP treatment enhanced the activity of antioxidant enzymes such as CAT, SOD and APX which are important to scavenge the over accumulation of ROS. SOD enzyme is known to convert superoxide ion into H2O2 which is eliminate by CAT [19] . Our results corroborate recent findings in green bell pepper where Cao et al. [17] monitored the effect of 1-MCP treatment on green bell pepper senescence during storage at 20˚C found that 1-MCP treatment increase the activity of antioxidant enzyme (APX, CAT and SOD) indirectly lowers the ROS content. In addition, 1-MCP treatment increases and lowers the antioxidant enzymes activities and MDA content respectively during the storage of broccoli [35] .
The result of our study also showed that, 1-MCP treatment significantly inhibits the activities of PAL and POD. PAL is known to be responsible for the synthesis of phenolic compounds, which then become substrates for oxidation enzymes such as PPO and POD. The higher amount of POD is reported to be involved in the chlorophyll degradation of horticultural products leading to their senescence [40] [41] . Therefore, we can suggest that the lower POD is probably related to the lack of substrate due to the inhibitory action of 1-MCP on PAL. Our results were in accordance of those of Salvador et al. [42] and Massolo et al. [43] where they respectively found that 1-MCP treatment reduced PAL activity in mandarin (“Nova” and “Ortanique”) and POD activity in eggplant.
5. Conclusion
Positive effects of 1-MCP were observed in this investigation on physicochemical quality of Z. latifolia during storage at 25˚C for 6 days. A postharvest application of 1-MCP significantly maintained the visual appearance of fresh Z. latifolia and retarded the increase of weight loss. In addition, the effect of 1-MCP treatment on delaying the senescence could be attributed to its capacity to retard the decrease of AsA content, improve the activities of antioxidant enzymes (APX, CAT and SOD) and inhibit the accumulation of ROS, MDA, thereby PLD, LOX, lipase as well as POD and PAL were inhibited. The shelf-life of Z. latifolia could prolong to 6 d during storage at 25˚C.
Acknowledgments
This work was funded by the research start-up funding from Nanjing Normal University (184080H202B117) and the general scientific research project of Zhejiang Provincial Department of Education (Y202147859).
NOTES
*Co-first authors.
#Corresponding author.