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Influence of drop shock on physiological responses and genes expression of apple fruit
Journal Pre-proofs Influence of drop shock on physiological responses and genes expression of apple fruit Fangxu Xu, Fei Lu, Zhigang Xiao, Zhe Li PII: DOI: Reference:
S0308-8146(19)31538-9 https://doi.org/10.1016/j.foodchem.2019.125424 FOCH 125424
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
30 April 2019 21 August 2019 22 August 2019
Please cite this article as: Xu, F., Lu, F., Xiao, Z., Li, Z., Influence of drop shock on physiological responses and genes expression of apple fruit, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.125424
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Influence of drop shock on physiological responses and genes expression of apple fruit
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Running title: Influence of shock on physiological responses of apple
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Fangxu Xu1 · Fei Lu2,* · Zhigang Xiao2 · Zhe Li2
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Abstract
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The aim of our study was to investigate the effect of drop shock on physiological responses and genes expression in
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harvested apple fruit stored at 20 ± 2 ℃. Ethylene production, respiratory rate, firmness, soluble solid content, relative
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electrical conductivity, LOX (lipoxygenase) activity, MDA (malondialdehyde) content, variation of volatile compounds,
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ethylene biosynthetic genes, and ethylene receptor genes of apple fruit were examined. The results indicated that drop
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shock observably resulted in the increase of ethylene production, respiratory rate, soluble solid content, relative
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electrical conductivity, LOX activity, MDA content and gene expression levels in apples. Furthermore, drop shock
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significantly decreased firmness and high-intensitive drop shock stimulated the accumulation of aldehydes and esters in
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harvested apples. Overall, the greater impact on apple quality is the effect of larger amplitude of shock during truck
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transportation, which seriously reduced storage life and quality of postharvest apples.
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Keywords Drop shock · Physiological responses · Volatile compounds · Genes expression · Apple
* Fei Lu [email protected] 1 Experimental Teaching Center, Shenyang Normal University, Shenyang, Liaoning, 110034, China 2 College of Grain Science and Technology, Shenyang Normal University, Shenyang, Liaoning, 110034, China 1
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1. Introduction
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Mechanical damage is the main factor causing the loss of fruits and vegetables after harvest. Mechanical damage
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not only causes sensory changes and nutrients loss, but also increases the risk of microbial infection and accelerates
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ripening thereby seriously affecting the commercial quality and economic benefit of postharvest fruits (Jung et al. 2018;
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Fu et al. 2018; Tasaki et al. 2019). In general, there are two types of mechanical damage in fruits. One is low stress
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fatigue damage caused by reciprocal action of vibration acceleration, which may result in the change of fruit structure.
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The other is the damage caused by shock, such as free fall or collision while packing. This kind of damage is mainly
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manifested as plastic deformation, and is usually disguised as mechanical damage (Lu et al. 2019; Xu and Liu 2017;
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Guo et al. 2013). A slight vibration has little effect on the quality of the apple fruit during actual truck transportation.
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The effect of slight vibration on some physiological characteristics can also be recovered after a period of stillness (Lu
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and Zhou 2009). In fact, the greater impact on apple quality is the effect of larger amplitude of shock during truck
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transportation (Mu et al. 2019; Chang et al. 2017; Lu et al. 2010).
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In recent years, a large number of studies have examined simulated vibration or shock test and damage mechanism
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during transportation, which provides an effective way to develop or improve the packaging technology of fruits
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(Tasaki et al. 2019; Shan and Xu 2015; Lin et al. 2014). For example, Van and De (2005) found mechanical damage
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was caused by the decomposition of cell wall components due to the action of cell wall related proteins that are caused
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by the physical damage of fruits. Lu and Zhou (2009) considered that the major reason of mechanical damage was
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fatigue vibration. They developed a mechanical damage model of fatigue vibration and a determination model
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parameters. Kang et al. (2003) explained the mechanism of damage during fruit transportation using Palmgren-Miner
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theory that the damage of fruit accumulated gradually and finally reached fatigue damage, thereby causing vibration
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damage. Vibration affects infiltration of epidermal cells and degradation of cell walls, which further affects the hardness
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and external variation of fruits (Zhou et al. 2007). However, Yang et al. (2002) indicated that mechanical vibration
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stimulated physiological reaction of fruits and moderate mechanical vibration frequency was helpful for callus growth.
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They also found that the membrane potential changed after mechanical vibration stimulation, which increased the
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ability of cells to absorb and transfer substances. Of course, temperature, maturity, moisture content and storage time
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also have different effects on mechanical damage of fruits. Klein (1987) considered the mechanical damage degree of
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fruits decreased with the prolonging of storage time, but Brusewitz and Bartsch (1989) suggested the change of
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collision energy per unit damage volume increased with the prolonging of storage period. Su et al. (2011) pointed out
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the tissue maturity of apples was also closely related to its callus ability after mechanical injury. The above results have
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important practical significance for reducing the loss of fruits and maintaining the quality of fruits. But the underlying
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mechanisms of internal changes in fruits after shock have not been clarified. So, it is significant to study the effect of
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shock on physiological and biochemical responses of fruits at the gene level.
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So far, there have been no reports on the influence of drop shock on physiological responses and genes expression
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in harvested apple fruit. The aim of our study is to discuss the effect of drop shock on commercial quality and ethylene
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biosynthetic genes and ethylene receptor genes expression in apples. The results provides a theoretical basis for
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designing a reasonable method to reduce the shock damage of fruits during transportation.
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2. Materials and Methods
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2.1. Sample Preparation and Treatments
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Mature apple fruit (Malus domestica Borkh. cv. Fuji) were harvested in a farm of Yingkou, China and rapidly
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transported to the lab. Scatheless apples of the same size (150 ± 50 g) and colour were selected for our test. Then, these
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apples were stochasticly separated into four groups of 3000 g each. The four groups were respectively dropped on four
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different contact surfaces (marble, high-density polyethylene (HDPE) plastics, corrugated board, and polystyrene
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foamed plastics) at 20 ± 2 ℃. The drop heights were chosen at 10 cm, 20 cm, 30 cm, 40 cm and 50 cm, respectively.
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After shocking, apples were stored for the subsequent test and the average values were reported.
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2.2. Measurement of Ethylene Production and Respiratory Rate
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Apples were sealed in a glass bottle (3 L) for 60 min. Then gas (1 mL) was extracted from the sealed glass bottle, 2
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and injected into a gas chromatograph (GC-2014C, Shimadzu, Japan). The temperature of column (30 m × 0.32 mm)
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was 50 ℃, the current velocity of N2 was 3.0 mL min–1 and the temperature of detector was 240 ℃.
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CO2 production was determined to express the respiration rate. Samples were sealed in a glass bottle (3 L) for 30
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min, and CO2 was collected to determine respiration rate with a portable infrared gas analyzer (Gas ID, Smiths
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Detection, USA).
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2.3. Measurement of Firmness and Soluble Solid Content
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Firmness was tested by a food texture analyzer (SMSTA.XTPlus, Stable Micro System, UK) with a 8 mm diameter probe at a penetration speed of 3 mm s−1. Four tests were randomly operated at different sides from each apple fruit. Homogenized apples were added aqua destillata (50 mL), then centrifuged for 5 min at 6,000 × g. The supernate
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was tested for soluble solid content (%) by a refractometer (MASTER-53S, ATAGO, Japan).
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2.4. Measurement of Relative Electrical Conductivity, LOX Activity and MDA Content
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Samples were cutted into slices of 2 mm and oscillated in ultrapure water for 20 min, whereafter, they were rinsed
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by ultrapure water. Values of electrical conductivity were measured before and after boiling for 20 min, and the specific
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value was the relative electrical conductivity.
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Sample (approx. 1 g) was prepared and homogenized with 5 mL precooling extraction buffer solution. Then the
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mixture was centrifuged at 4,000 × g for 30 min and the supernatant was the crude enzyme. A 200 μL of crude enzyme
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was added in 3 mL acetic acid-sodium acetate buffer and 50 μL sodium linoleate solution (0.001 mol L−1) after warm
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bath at 30 ℃ for 10 min. Afterwards, absorbances of supernatant at 234 nm were measured by a spectrophotometer
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(UV-4802, Unico, China) for LOX activity.
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Homogenized apples (approx. 2 g) were added in thiobarbituric acid (2 mL, 0.6%) for blending, whereafter, the
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mixture were centrifuged at 4,000 × g for 10 min. Samples were thermal treated for 20 min, then centrifuged again at
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4,000 × g for 10 min. Absorbances at 450 and 532 nm were measured and the malondialdehyde (MDA) content was
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computed as: MDA (nmol g -1 ) = 6450 A 532 - 560 A 450 .
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2.5. Measurement of Volatile Compounds
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volatile compounds were assayed by a portable electronic nose (PEN 3, Airsense, Germany) equipped with 10
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metal oxide sensors array (Table 3). Apples (10 g) were placed in a beaker and sealed with polypropylene film for 5
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min at room temperature. Then headspace suction method was used for the data collection. The measurement
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parameters were as follows: sensor cleaning time 220 s; automatic zero time setting 10 s; sample preparation time 5 s;
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sample test time 50 s; sample determination interval 1 s; internal flow 100 mL min–1; sample flow 100 mL min–1. The
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sensor response is given by the ratio of the conductivity response of the sensors to the sample gas (G) relative to the
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carrier gas (G0) over time (G/G0).
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2.6. RNA Isolation and cDNA Preparation
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RNA in apples was extracted referring to the way of modified Trizol. Reverse transcription was handled according
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to the operating instructions of TIANScript RT kit (Tiangen, Beijing, China).
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2.7. Simple RT-PCR Analysis
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The design of primers was operated using Primer Premier 5.0 software. The synthesis of primers was carried out
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by Kingsry Biotechnology Co. Ltd. (Table 1).
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2.8. Statistical Analysis
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Statistical analysis were executed by analysis of variance in SPSS software. Significant differences were analyzed
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by Tukey’s test at the 5% level. All experimental results were showed as the mean ± SE.
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3. Results and Discussion
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3.1. Effect of Different Shock Conditions on Ethylene Production and Respiratory Rate
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As a plant hormone, ethylene has the physiological effect of enhancing respiration rate and promoting fruit
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ripening. If physical stimulation is given, ethylene production will increase significantly (Xu and Liu 2017; Fu et al.
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2018). As shown in Fig. 1a and b, the ethylene production and respiratory rate in samples increasingly went up with the 3
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increase of shock height, and reached the peak at the shock height of 30 cm. Our present results suggested ethylene
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production and respiratory rate of apples decreased if the shock height continued to increase, which indicated that
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damaged apples still had metabolic activity and repeated self-regulation to achieve balance. Moreover, different contact
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surfaces also had different effects on ethylene production and respiratory rate of fruits. As shown in Fig. 1a and b,
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marble had the most significant effect (p < 0.05) on ethylene production and respiratory rate compared to the other three
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materials. Robitaille and Janick (1973) found that external injury accelerated ethylene production of apples. Wu et al.
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(2012) also indicated that mechanical damages made an significant increase of ethylene production and respiratory rate
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in apple fruit during the later period of storage, which was similar with our present results. The possible reason was that
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the tissue structure on the surface of fruit and the regionalization inside cells were severely damaged after shock,
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thereby significantly increasing the oxygen supply in the tissues. Then the contact opportunities of substrates and
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enzymes were increased that leading to the enhance of respiration. Up to now, there was no reports related to the effect
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of shock height on ethylene production and respiratory rate in apple fruit.
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3.2. Effect of Different Shock Conditions on Firmness and Soluble Solid Content
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Firmness of fruits refers to the resistance of the pulp under shock. Firmness depends on the amount of pectin in the
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cell walls of pulp (Fu et al. 2017). Fig. 1c showed that firmness of apple fruit gradually declined with the increase of
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shock height. This was mainly because the impact force caused by the shock height gravely hurt the apple fruit.
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Furthermore, different contact surfaces had different effects on the descent degree of firmness in apples. Foamed
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plastics had the least effect on firmness, while marble had the significantly effect (p < 0.05) on that of apple fruit. Besides, the cushioning effects of each materials were various. Soft contact materials showed a better cushioning effect. Thus it can be seen that the hardness of contact surfaces seriously affected the firmness of apple fruit. Similar results were reported by Zhang et al. (2018) and Zhou and Li (2007), who observed mechanical damages accelerated softening of grape and Huanghua pears. These results indicated that mechanical damage destroyed the tissue structure of the cell walls, which rapidly promoted fruit softening and physiological metabolism. Soluble solids are the general term for all water-soluble compounds in liquid or liquid foods. Damaged fruits may produce large amounts of polysaccharides and polyphenols to make up for the lost components (Shan and Xu 2015). As shown in Fig. 1d, the soluble solid content in apple fruit gradually increased with the increase of shock height, and reached the peak value at the shock height of 30 cm. As the shock height continued to rise, the soluble solid content decreased inversely. Certainly, different contact materials had different effects on soluble solid content in apples. In
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these four contact materials, marble had the prominent effect (p < 0.05) on soluble solid content of apple fruit.
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Analogous results was found by Lu et al. (2019) in our preliminary experiment, which showed that mechanical damage
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during truck transportation increased soluble solid content compared to the control group. Jung et al. (2018) observed
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the decrement of soluble solid content in grape suffered from vibration was 1.3° Brix as compared to the control grape
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group. Mu et al. (2019) found that the effect of mechanical damage on storage quality of spinach was obvious,
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especially on the content of soluble solids. Overall it can be concluded that mechanical damage caused serious injure to
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harvested apple fruit, especially at high hardness contact materials.
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3.3. Effect of Different Shock Conditions on Relative Electrical Conductivity, LOX Activity and MDA Content
125 126 127 128 129 130 131 132 133
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Relative electrical conductivity indicates the membrane permeability of fruit (Huang et al. 2015). As shown in
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Table 2, relative electrical conductivity of apple fruit gradually went up with the increase of shock height compared
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with the control group, and reached the peak value at the shock height of 30 cm. Moreover, the influence of contact
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materials on relative electrical conductivity was also obvious. The relative electrical conductivity of apples shocked on
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marble was 24.8 % higher than that of shocked on foamed plastics at the shock height of 30 cm, which indicated
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remarkable differences (p < 0.05) were found among the four contact materials. Huang et al. (2015) found relative
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electrical conductivity of strawberries treated with anti-vibration package was 20.2 % lower than that of treated with
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traditional package. Chang et al. (2017) also indicated anti-vibration package treatment significantly inhibited the
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increase of the relative electrical conductivity, thereby reduced the damage of pulp tissue in kiwifruit. The above results
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might be due to the membrane damage that caused by shock during transport, which leading to the increase of cell 4
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leakage.
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Superabundant lipoxygenase (LOX) activity in fruit may decrease nutritional quality and increased difficulty of
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storage (Tasaki et al. 2019). As shown in Table 2, LOX activity in samples went up gradually with the increase of shock
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height compared to the control group. The peak of LOX activity was obtained at the shock height of 50 cm, and the
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difference reached a significant level (p < 0.05) in comparison with the control group. Of course, the effect of contact
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materials on LOX activity was especially significant. Table 2 showed that marble with higher hardness had the most
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obvious effect on LOX activity in apple fruit. In our study, the best anti-shock package material was foamed plastics,
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which had the minimal effect on LOX activity of samples. Therefore, soft materials should be used as far as possible
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during the fruit transportation. Shen et al. (1999) also considered mechanical damage promoted the increase of LOX
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activity in apples that caused the aging character to appear in advance.
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Malondialdehyde (MDA) is one of the main products of membrane lipid peroxidation, which can further damage
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the biofilm. MDA content can be used to reflect the response of plant cells to adverse conditions, and is an important
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indicator of membrane lipid peroxidation (Xu et al. 2019). As shown in Table 2, MDA content in apples increased
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slowly during the shock height of 0-30 cm, and reached the peak value at the shock height of 30 cm, then decreased
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gradually during the shock height of 30-50 cm. The probable reason was the fruits can defend itself and maintain a
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relatively balanced state when subjected to external stress. Besides, MDA content in samples shocked on marble was
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70.6 % higher than that of shocked on foamed plastics at the shock height of 30 cm, which suggested that observable
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differences (p < 0.05) were obtained among the four contact materials. These results were extremely consistent with our
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earlier results (Lu et al. 2019), which considered the greater impact on apple quality was the effect of larger amplitude
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of shock during truck transportation. Similar results were found by Zeng et al. (2016) who suggested mechanical injury
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accelerated MDA production and membrane lipid peroxidation. Shen et al. (1999) reported MDA in damaged apples
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accumulated largely, which indicated mechanical damage destroyed the integrity of the membrane.
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3.4. Effect of Different Shock Conditions on Variation of Volatile Compounds
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Table 4 showed the effects of different shock conditions on variation of volatile compounds in apples. A total of 30
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volatile compounds were identified and quantified from apples, and the major volatile compounds were alcohols,
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aldehydes and esters. In addition, the relative contents of 2-methylbutyl acetate, hexyl acetate, 2-hexenal, hexanal and
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butyl acetate were higher in all volatile compounds, as shown by Lu et al. (2018). As shown in Table 4, the effects of
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different drop shock conditions on variation of volatile compounds in apples were different. In general, aldehydes and
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esters in samples shocked on marble were relatively higher than that of other contact material. This demonstrated that
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high-intensitive drop shock stimulated the accumulation of aldehydes and esters. However, as for alcohols, the results
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were various. The possible reason was that mechanical damage promoted fruit physiological metabolism, resulting in
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the accumulation of aldehydes and esters. Up to now, there were few research results on the effects of mechanical
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damage on the changes of volatile compounds in fruit.
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3.5. Effect of Different Shock Conditions on Ethylene Biosynthetic Genes
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As a plant gas hormone, ethylene is involved in the regulation of many important processes in plants. ACS and
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ACO are two key enzymes for ethylene biosynthesis, which has significant meanings for the regulation of ripening and
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senescence of fruits (Xu et al. 2016 a). As shown in Fig. 2, the gene expression levels of MdACS1 and MdACO1 in
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apple fruit enhanced slowly when the shock height was 0-30 cm, and then weakened gradually with the increase of
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shock height. This trend was consistent with the above results of ethylene production and respiratory rate. The
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expression of MdACS1 and MdACO1 in samples shocked on marble at the height of 30 cm was over 10 fold higher than
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that of untreated fruit. The other contact materials similarly enhanced the expression levels of ethylene biosynthetic
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genes, although not as prominent as the effect of marble. Therefore, it was proved that reducing the impact in
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transportation was of great significance for prolonging storage life and improving commercial quality of fruit in theory.
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3.6. Effect of Different Shock Conditions on Ethylene Receptor Genes
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Ethylene physiological action is finally completed through ethylene receptors and its signal transduction (Xu et al.
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2016 b). As shown in Fig. 2, different shock treatment effectively improved the expression levels of ethylene receptor 5
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genes in varying degree compared with the untreated apples. However, the enhanced effect of shock treatment of
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marble on the expression of MdETR1 and MdCTR1 was the most obvious (p < 0.05) comparing to the other contact
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materials. Furthermore, the effect of shock height on the expression of ethylene receptor gene was significant. Fig. 2
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indicated that ethylene receptor gene expression reached the peak at the shock height of 30 cm, which was significant (p
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< 0.05) compared with the control group. The possible reason for the decline in gene expression levels of ethylene
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receptor with the increase of shock height was the repair and regulation of the fruit itself. The variation trend of
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ethylene receptor gene expression was similar to the above results of ethylene biosynthetic genes, which indicated that
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external damage seriously affected the ethylene signal transduction pathway of fruits. Thus it can be seen that
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anti-shock package was necessary to reduce mechanical damage of fruits caused by vibration or shock.
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4. Conclusions
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Our present study investigated the influence of drop shock on physiological responses and ethylene biosynthetic
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genes and ethylene receptor genes expression in harvested apple fruit. The results suggested drop shock dramatically
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increased ethylene production, respiratory rate, soluble solid content, relative electrical conductivity, LOX activity,
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MDA content and gene expression levels of MdACS1, MdACO1, MdETR1 and MdCTR1 in apple fruit. Furthermore,
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firmness of harvested apples declined evidently after drop shock. High-intensitive drop shock stimulated the
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accumulation of aldehydes and esters in apples. In conclusion, drop shock resulted in serious deterioration of fruit
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quality and it is especially necessary to search a scientific and effective anti-shock measure. Further research has been
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undertaken to examine changes of related proteins in fruits after drop shock with proteomics, which aims to look for the
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underlying mechanisms of internal changes in fruits after drop shock.
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Acknowledgements This work was supported by National Natural Science Foundation of China (31201434) and
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Natural Science Foundation of Liaoning Province (20180550837).
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Table 1 Sequences of primers and probes for quantitative analysis of genes expression with real-time PCR. Name
Accession number
Product size
Sequence
MdACS1
U73815
146
MdACO1
DQ137850
129
MdETR1
DQ137848
118
MdCTR1
AY670703
159
18s rRNA
CV82635
87
5′TGAAATCTATGCAGCAACCGGCC3′ 3′CAACCCTAAATCCAGGGAAC5′ 5′ACCTTCCTTCCTCAAACAT3′ 3′CACCTCAATCTGGTCACCTAAG5′ 5′TGTTGGGAATGCTGTGAAGT3′ 3′ATGATTTGCTTGCAGCTTGG5′ 5′AACCTCCCATTGTTCATCGT3′ 3′CTTCTGGTGGTGCCATCCATTCA5′ 5′GGGTTCGATTCCGGAGAGG3′ 3′CCGTGTCAGGATTGGGTAAT5′
293 294 295 296 297 298
Table 2 Effects of different shock conditions on electrical conductivity, LOX activity and MDA content of apple fruit at 20 ± 2 ℃. Values are mean ± SE of triplicate samples. Drop height (cm)
Measuring item Shock treatment Relative electrical conductivity (%)
0
10
20
30
Marble
34.1±0.02Aa
46.5±0.15Ab
57.2±0.26Ac
71.5±0.35Ad
65.5±0.24
HDPE plastics
34.1±0.02Aa
40.2±0.13Bb
55.2±0.35Ac
65.1±0.48Bd
59.4±0.26
Corrugated board
34.1±0.02Aa
36.3±0.09Cb
42.5±0.54Bc
61.5±0.25Cd
55.1±0.05
Foamed plastics
34.1±0.02Aa
34.5±0.05Ca
45.0±0.15Bb
57.3±0.19Dc
51.2±0.02
90±0.25Aa
159±0.91Ab
164±0.36Ac
181±0.78Ad
179±0.51A
90±0.25Aa
140±0.84Bb
143±0.54Bb
160±0.59Bc
169±0.31B
90±0.25Aa
118±0.58Cb
125±0.57Cc
150±0.93Cd
148±0.57C
Foamed plastics
90±0.25Aa
99±0.72Db
110±0.86Dc
119±0.58Dd
115±0.84D
Marble
2.2±0.03Aa
4.2±0.05Ab
5.5±0.03Ac
5.8±0.28Ac
5.2±0.21A
2.2±0.03Aa
3.5±0.02Bb
4.5±0.05Bc
4.6±0.16Bc
4.1±0.06B
2.2±0.03Aa
3.1±0.16Bb
3.7±0.16Cc
4.2±0.24Bd
2.4±0.08C
2.2±0.03Aa
2.3±0.09Ca
2.4±0.49Da
3.4±0.38Cb
1.5±0.27D
Marble LOX activity HDPE plastics Corrugated board (min g–1)
MDA content HDPE plastics (nmol g–1) Corrugated board Foamed plastics 299
a
300
b
All means in the same column followed by different letters (A-C) are significantly (p <0.05) different. All means in the same row followed by different letters (a-d) are significantly (p <0.05) different.
301 302
Table 3 Standard sensor arrays and performance specification in electronic nose PEN 3. Number
Sensor
Performance
Notes
S1
W1C
Aromatic
Methylbenzene, 10 ppm
S2
W5S
Broadrange
NO2, 1 ppm
S3
W3C
Ammonium hydroxide
Benzene, 10 ppm
S4
W6S
Hydrogen
H2, 100 ppm
S5
W5C
Aromatic aliphatics
Dimethylmethane, 1 ppm
S6
W1S
Broad methane
CH3, 100 ppm 8
40
S7
W1W
Sulfur organic
H2S, 1 ppm
S8
W2S
Broad alcohol
CO, 100 ppm
S9
W2W
Sulfurchlorinate
H2S, 1 ppm
S10
W3S
Methane aliphatics
CH3, 100 ppm
303 304 305
Table 4 Effects of different shock conditions on variation of volatile compounds in apples.
9
306 307 308
Relative content (%)
unds
0 cm
ol
l
ate
anoate
tanoate
noate
309
50 cm
Marble
HDPE plastics
Corrugated board
Foamed plastics
Marble
HDPE plastics
Corrugated board
0.69 a
0.58 a
0.57 a
0.44 a
0.75 a
0.61 a
0.65 a
0.19 a
0.36 b
0.22 a
0.31 b
0.15 a
0.20 a
0.19 a
23.65 a
25.85 a
26.36 ab
29.65 b
19.63 c
20.58 ac
22.16 ac
1.54 a
1.37 a
1.29 a
1.21 a
1.93 a
1.77 a
1.62 a
-
-
-
0.02 a
-
-
0.01 a
24.16 a
22.57 a
21.69 a
20.60 a
26.96 a
24.48 a
23.96 a
0.29 ab
0.45 a
0.48 a
0.52 a
0.12 b
0.31 ab
0.28 ab
0.89 a
0.62 a
0.58 a
0.49 a
1.05 a
0.85 a
0.79 a
0.15 a
0.26 a
0.21 a
0.31 a
0.10 a
0.19 a
0.17 a
98.26 a
80.16 b
79.47 b
75.25 b
100.78 a
92.09 a
89.65 a
112.69 a
100.15 b
102.36 b
100.23 b
125.65 a
117.28 a
118.52 a
0.48 a
0.39 a
0.33 a
0.29 a
0.65 a
0.41 a
0.40 a
0.84 a
0.69 a
0.65 a
0.52 a
0.96 a
0.71 a
0.68 a
359.69 a
315.25 b
302.63 b
256.32 c
405.20 d
365.69 a
358.85 a
89.66 a
72.65 b
70.19 b
70.20 b
98.36 a
80.11 a
79.52 a
22.65 a
15.29 b
16.08 b
11.27 b
29.85 a
18.66 b
17.98 b
223.65 a
179.69 b
185.46 b
156.35 c
256.93 a
201.58 a
198.64 b
41.12 a
35.26 ab
34.98 ab
32.02 b
45.21 a
38.55 ab
37.89 ab
1.20 a
0.89 a
0.95 a
0.56 a
1.45 a
1.02 a
0.98 a
8.36 a
4.22 b
3.98 b
2.21 c
10.03 d
6.32 b
7.05 a
29.65 a
23.55 a
24.08 a
21.26 a
32.16 a
24.56 a
25.05 a
5.96 a
4.45 b
5.08 a
1.59 b
7.66 a
5.95 a
6.09 a
2.69 a
1.53 b
1.66 b
1.25 b
3.44 a
2.58 a
2.36 a
15.66 a
12.85 ab
13.09 a
10.36 b
17.56 c
14.26 a
13.18 a
1.27 a
0.96 a
0.85 a
0.63 a
2.59 b
1.58 a
1.67 a
8.21 a
7.52 a
6.36 ab
5.20 b
9.85 a
7.92 a
8.05 a
41.56 a
35.28 b
33.19 b
30.26 b
45.25 a
41.52 a
40.05 a
22.65 a
20.05 a
19.98 ab
18.24 b
25.23 a
22.69 a
21.59 ab
5.39 a
3.06 b
3.18 b
2.56 b
7.85 c
4.32 b
5.68 a
15.87 a
12.69 b
10.23 c
9.68 c
18.82 d
14.45 a
14.98 a
Declaration of interests
310 10
311 312
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
313 314 315
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
316
317 318 319 320 321 322
Highlights • Drop shock observably increased ethylene production and respiratory rate. • Drop shock enhanced LOX activity and MDA content . • Drop shock effectively improved gene expression levels.
323 324 325
11
S0308-8146(19)31538-9 https://doi.org/10.1016/j.foodchem.2019.125424 FOCH 125424
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
30 April 2019 21 August 2019 22 August 2019
Please cite this article as: Xu, F., Lu, F., Xiao, Z., Li, Z., Influence of drop shock on physiological responses and genes expression of apple fruit, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.125424
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© 2019 Published by Elsevier Ltd.
1
Influence of drop shock on physiological responses and genes expression of apple fruit
2
Running title: Influence of shock on physiological responses of apple
3
Fangxu Xu1 · Fei Lu2,* · Zhigang Xiao2 · Zhe Li2
4
Abstract
5
The aim of our study was to investigate the effect of drop shock on physiological responses and genes expression in
6
harvested apple fruit stored at 20 ± 2 ℃. Ethylene production, respiratory rate, firmness, soluble solid content, relative
7
electrical conductivity, LOX (lipoxygenase) activity, MDA (malondialdehyde) content, variation of volatile compounds,
8
ethylene biosynthetic genes, and ethylene receptor genes of apple fruit were examined. The results indicated that drop
9
shock observably resulted in the increase of ethylene production, respiratory rate, soluble solid content, relative
10
electrical conductivity, LOX activity, MDA content and gene expression levels in apples. Furthermore, drop shock
11
significantly decreased firmness and high-intensitive drop shock stimulated the accumulation of aldehydes and esters in
12
harvested apples. Overall, the greater impact on apple quality is the effect of larger amplitude of shock during truck
13
transportation, which seriously reduced storage life and quality of postharvest apples.
14 15
Keywords Drop shock · Physiological responses · Volatile compounds · Genes expression · Apple
* Fei Lu [email protected] 1 Experimental Teaching Center, Shenyang Normal University, Shenyang, Liaoning, 110034, China 2 College of Grain Science and Technology, Shenyang Normal University, Shenyang, Liaoning, 110034, China 1
16
1. Introduction
17
Mechanical damage is the main factor causing the loss of fruits and vegetables after harvest. Mechanical damage
18
not only causes sensory changes and nutrients loss, but also increases the risk of microbial infection and accelerates
19
ripening thereby seriously affecting the commercial quality and economic benefit of postharvest fruits (Jung et al. 2018;
20
Fu et al. 2018; Tasaki et al. 2019). In general, there are two types of mechanical damage in fruits. One is low stress
21
fatigue damage caused by reciprocal action of vibration acceleration, which may result in the change of fruit structure.
22
The other is the damage caused by shock, such as free fall or collision while packing. This kind of damage is mainly
23
manifested as plastic deformation, and is usually disguised as mechanical damage (Lu et al. 2019; Xu and Liu 2017;
24
Guo et al. 2013). A slight vibration has little effect on the quality of the apple fruit during actual truck transportation.
25
The effect of slight vibration on some physiological characteristics can also be recovered after a period of stillness (Lu
26
and Zhou 2009). In fact, the greater impact on apple quality is the effect of larger amplitude of shock during truck
27
transportation (Mu et al. 2019; Chang et al. 2017; Lu et al. 2010).
28
In recent years, a large number of studies have examined simulated vibration or shock test and damage mechanism
29
during transportation, which provides an effective way to develop or improve the packaging technology of fruits
30
(Tasaki et al. 2019; Shan and Xu 2015; Lin et al. 2014). For example, Van and De (2005) found mechanical damage
31
was caused by the decomposition of cell wall components due to the action of cell wall related proteins that are caused
32
by the physical damage of fruits. Lu and Zhou (2009) considered that the major reason of mechanical damage was
33
fatigue vibration. They developed a mechanical damage model of fatigue vibration and a determination model
34
parameters. Kang et al. (2003) explained the mechanism of damage during fruit transportation using Palmgren-Miner
35
theory that the damage of fruit accumulated gradually and finally reached fatigue damage, thereby causing vibration
36
damage. Vibration affects infiltration of epidermal cells and degradation of cell walls, which further affects the hardness
37
and external variation of fruits (Zhou et al. 2007). However, Yang et al. (2002) indicated that mechanical vibration
38
stimulated physiological reaction of fruits and moderate mechanical vibration frequency was helpful for callus growth.
39
They also found that the membrane potential changed after mechanical vibration stimulation, which increased the
40
ability of cells to absorb and transfer substances. Of course, temperature, maturity, moisture content and storage time
41
also have different effects on mechanical damage of fruits. Klein (1987) considered the mechanical damage degree of
42
fruits decreased with the prolonging of storage time, but Brusewitz and Bartsch (1989) suggested the change of
43
collision energy per unit damage volume increased with the prolonging of storage period. Su et al. (2011) pointed out
44
the tissue maturity of apples was also closely related to its callus ability after mechanical injury. The above results have
45
important practical significance for reducing the loss of fruits and maintaining the quality of fruits. But the underlying
46
mechanisms of internal changes in fruits after shock have not been clarified. So, it is significant to study the effect of
47
shock on physiological and biochemical responses of fruits at the gene level.
48
So far, there have been no reports on the influence of drop shock on physiological responses and genes expression
49
in harvested apple fruit. The aim of our study is to discuss the effect of drop shock on commercial quality and ethylene
50
biosynthetic genes and ethylene receptor genes expression in apples. The results provides a theoretical basis for
51
designing a reasonable method to reduce the shock damage of fruits during transportation.
52
2. Materials and Methods
53
2.1. Sample Preparation and Treatments
54
Mature apple fruit (Malus domestica Borkh. cv. Fuji) were harvested in a farm of Yingkou, China and rapidly
55
transported to the lab. Scatheless apples of the same size (150 ± 50 g) and colour were selected for our test. Then, these
56
apples were stochasticly separated into four groups of 3000 g each. The four groups were respectively dropped on four
57
different contact surfaces (marble, high-density polyethylene (HDPE) plastics, corrugated board, and polystyrene
58
foamed plastics) at 20 ± 2 ℃. The drop heights were chosen at 10 cm, 20 cm, 30 cm, 40 cm and 50 cm, respectively.
59
After shocking, apples were stored for the subsequent test and the average values were reported.
60
2.2. Measurement of Ethylene Production and Respiratory Rate
61
Apples were sealed in a glass bottle (3 L) for 60 min. Then gas (1 mL) was extracted from the sealed glass bottle, 2
62
and injected into a gas chromatograph (GC-2014C, Shimadzu, Japan). The temperature of column (30 m × 0.32 mm)
63
was 50 ℃, the current velocity of N2 was 3.0 mL min–1 and the temperature of detector was 240 ℃.
64
CO2 production was determined to express the respiration rate. Samples were sealed in a glass bottle (3 L) for 30
65
min, and CO2 was collected to determine respiration rate with a portable infrared gas analyzer (Gas ID, Smiths
66
Detection, USA).
67
2.3. Measurement of Firmness and Soluble Solid Content
68 69 70
Firmness was tested by a food texture analyzer (SMSTA.XTPlus, Stable Micro System, UK) with a 8 mm diameter probe at a penetration speed of 3 mm s−1. Four tests were randomly operated at different sides from each apple fruit. Homogenized apples were added aqua destillata (50 mL), then centrifuged for 5 min at 6,000 × g. The supernate
71
was tested for soluble solid content (%) by a refractometer (MASTER-53S, ATAGO, Japan).
72
2.4. Measurement of Relative Electrical Conductivity, LOX Activity and MDA Content
73
Samples were cutted into slices of 2 mm and oscillated in ultrapure water for 20 min, whereafter, they were rinsed
74
by ultrapure water. Values of electrical conductivity were measured before and after boiling for 20 min, and the specific
75
value was the relative electrical conductivity.
76
Sample (approx. 1 g) was prepared and homogenized with 5 mL precooling extraction buffer solution. Then the
77
mixture was centrifuged at 4,000 × g for 30 min and the supernatant was the crude enzyme. A 200 μL of crude enzyme
78
was added in 3 mL acetic acid-sodium acetate buffer and 50 μL sodium linoleate solution (0.001 mol L−1) after warm
79
bath at 30 ℃ for 10 min. Afterwards, absorbances of supernatant at 234 nm were measured by a spectrophotometer
80
(UV-4802, Unico, China) for LOX activity.
81
Homogenized apples (approx. 2 g) were added in thiobarbituric acid (2 mL, 0.6%) for blending, whereafter, the
82
mixture were centrifuged at 4,000 × g for 10 min. Samples were thermal treated for 20 min, then centrifuged again at
83
4,000 × g for 10 min. Absorbances at 450 and 532 nm were measured and the malondialdehyde (MDA) content was
84
computed as: MDA (nmol g -1 ) = 6450 A 532 - 560 A 450 .
85
2.5. Measurement of Volatile Compounds
86
volatile compounds were assayed by a portable electronic nose (PEN 3, Airsense, Germany) equipped with 10
87
metal oxide sensors array (Table 3). Apples (10 g) were placed in a beaker and sealed with polypropylene film for 5
88
min at room temperature. Then headspace suction method was used for the data collection. The measurement
89
parameters were as follows: sensor cleaning time 220 s; automatic zero time setting 10 s; sample preparation time 5 s;
90
sample test time 50 s; sample determination interval 1 s; internal flow 100 mL min–1; sample flow 100 mL min–1. The
91
sensor response is given by the ratio of the conductivity response of the sensors to the sample gas (G) relative to the
92
carrier gas (G0) over time (G/G0).
93
2.6. RNA Isolation and cDNA Preparation
94
RNA in apples was extracted referring to the way of modified Trizol. Reverse transcription was handled according
95
to the operating instructions of TIANScript RT kit (Tiangen, Beijing, China).
96
2.7. Simple RT-PCR Analysis
97
The design of primers was operated using Primer Premier 5.0 software. The synthesis of primers was carried out
98
by Kingsry Biotechnology Co. Ltd. (Table 1).
99
2.8. Statistical Analysis
100
Statistical analysis were executed by analysis of variance in SPSS software. Significant differences were analyzed
101
by Tukey’s test at the 5% level. All experimental results were showed as the mean ± SE.
102
3. Results and Discussion
103
3.1. Effect of Different Shock Conditions on Ethylene Production and Respiratory Rate
104
As a plant hormone, ethylene has the physiological effect of enhancing respiration rate and promoting fruit
105
ripening. If physical stimulation is given, ethylene production will increase significantly (Xu and Liu 2017; Fu et al.
106
2018). As shown in Fig. 1a and b, the ethylene production and respiratory rate in samples increasingly went up with the 3
107
increase of shock height, and reached the peak at the shock height of 30 cm. Our present results suggested ethylene
108
production and respiratory rate of apples decreased if the shock height continued to increase, which indicated that
109
damaged apples still had metabolic activity and repeated self-regulation to achieve balance. Moreover, different contact
110
surfaces also had different effects on ethylene production and respiratory rate of fruits. As shown in Fig. 1a and b,
111
marble had the most significant effect (p < 0.05) on ethylene production and respiratory rate compared to the other three
112
materials. Robitaille and Janick (1973) found that external injury accelerated ethylene production of apples. Wu et al.
113
(2012) also indicated that mechanical damages made an significant increase of ethylene production and respiratory rate
114
in apple fruit during the later period of storage, which was similar with our present results. The possible reason was that
115
the tissue structure on the surface of fruit and the regionalization inside cells were severely damaged after shock,
116
thereby significantly increasing the oxygen supply in the tissues. Then the contact opportunities of substrates and
117
enzymes were increased that leading to the enhance of respiration. Up to now, there was no reports related to the effect
118
of shock height on ethylene production and respiratory rate in apple fruit.
119
3.2. Effect of Different Shock Conditions on Firmness and Soluble Solid Content
120
Firmness of fruits refers to the resistance of the pulp under shock. Firmness depends on the amount of pectin in the
121
cell walls of pulp (Fu et al. 2017). Fig. 1c showed that firmness of apple fruit gradually declined with the increase of
122
shock height. This was mainly because the impact force caused by the shock height gravely hurt the apple fruit.
123
Furthermore, different contact surfaces had different effects on the descent degree of firmness in apples. Foamed
124
134
plastics had the least effect on firmness, while marble had the significantly effect (p < 0.05) on that of apple fruit. Besides, the cushioning effects of each materials were various. Soft contact materials showed a better cushioning effect. Thus it can be seen that the hardness of contact surfaces seriously affected the firmness of apple fruit. Similar results were reported by Zhang et al. (2018) and Zhou and Li (2007), who observed mechanical damages accelerated softening of grape and Huanghua pears. These results indicated that mechanical damage destroyed the tissue structure of the cell walls, which rapidly promoted fruit softening and physiological metabolism. Soluble solids are the general term for all water-soluble compounds in liquid or liquid foods. Damaged fruits may produce large amounts of polysaccharides and polyphenols to make up for the lost components (Shan and Xu 2015). As shown in Fig. 1d, the soluble solid content in apple fruit gradually increased with the increase of shock height, and reached the peak value at the shock height of 30 cm. As the shock height continued to rise, the soluble solid content decreased inversely. Certainly, different contact materials had different effects on soluble solid content in apples. In
135
these four contact materials, marble had the prominent effect (p < 0.05) on soluble solid content of apple fruit.
136
Analogous results was found by Lu et al. (2019) in our preliminary experiment, which showed that mechanical damage
137
during truck transportation increased soluble solid content compared to the control group. Jung et al. (2018) observed
138
the decrement of soluble solid content in grape suffered from vibration was 1.3° Brix as compared to the control grape
139
group. Mu et al. (2019) found that the effect of mechanical damage on storage quality of spinach was obvious,
140
especially on the content of soluble solids. Overall it can be concluded that mechanical damage caused serious injure to
141
harvested apple fruit, especially at high hardness contact materials.
142
3.3. Effect of Different Shock Conditions on Relative Electrical Conductivity, LOX Activity and MDA Content
125 126 127 128 129 130 131 132 133
143
Relative electrical conductivity indicates the membrane permeability of fruit (Huang et al. 2015). As shown in
144
Table 2, relative electrical conductivity of apple fruit gradually went up with the increase of shock height compared
145
with the control group, and reached the peak value at the shock height of 30 cm. Moreover, the influence of contact
146
materials on relative electrical conductivity was also obvious. The relative electrical conductivity of apples shocked on
147
marble was 24.8 % higher than that of shocked on foamed plastics at the shock height of 30 cm, which indicated
148
remarkable differences (p < 0.05) were found among the four contact materials. Huang et al. (2015) found relative
149
electrical conductivity of strawberries treated with anti-vibration package was 20.2 % lower than that of treated with
150
traditional package. Chang et al. (2017) also indicated anti-vibration package treatment significantly inhibited the
151
increase of the relative electrical conductivity, thereby reduced the damage of pulp tissue in kiwifruit. The above results
152
might be due to the membrane damage that caused by shock during transport, which leading to the increase of cell 4
153
leakage.
154
Superabundant lipoxygenase (LOX) activity in fruit may decrease nutritional quality and increased difficulty of
155
storage (Tasaki et al. 2019). As shown in Table 2, LOX activity in samples went up gradually with the increase of shock
156
height compared to the control group. The peak of LOX activity was obtained at the shock height of 50 cm, and the
157
difference reached a significant level (p < 0.05) in comparison with the control group. Of course, the effect of contact
158
materials on LOX activity was especially significant. Table 2 showed that marble with higher hardness had the most
159
obvious effect on LOX activity in apple fruit. In our study, the best anti-shock package material was foamed plastics,
160
which had the minimal effect on LOX activity of samples. Therefore, soft materials should be used as far as possible
161
during the fruit transportation. Shen et al. (1999) also considered mechanical damage promoted the increase of LOX
162
activity in apples that caused the aging character to appear in advance.
163
Malondialdehyde (MDA) is one of the main products of membrane lipid peroxidation, which can further damage
164
the biofilm. MDA content can be used to reflect the response of plant cells to adverse conditions, and is an important
165
indicator of membrane lipid peroxidation (Xu et al. 2019). As shown in Table 2, MDA content in apples increased
166
slowly during the shock height of 0-30 cm, and reached the peak value at the shock height of 30 cm, then decreased
167
gradually during the shock height of 30-50 cm. The probable reason was the fruits can defend itself and maintain a
168
relatively balanced state when subjected to external stress. Besides, MDA content in samples shocked on marble was
169
70.6 % higher than that of shocked on foamed plastics at the shock height of 30 cm, which suggested that observable
170
differences (p < 0.05) were obtained among the four contact materials. These results were extremely consistent with our
171
earlier results (Lu et al. 2019), which considered the greater impact on apple quality was the effect of larger amplitude
172
of shock during truck transportation. Similar results were found by Zeng et al. (2016) who suggested mechanical injury
173
accelerated MDA production and membrane lipid peroxidation. Shen et al. (1999) reported MDA in damaged apples
174
accumulated largely, which indicated mechanical damage destroyed the integrity of the membrane.
175
3.4. Effect of Different Shock Conditions on Variation of Volatile Compounds
176
Table 4 showed the effects of different shock conditions on variation of volatile compounds in apples. A total of 30
177
volatile compounds were identified and quantified from apples, and the major volatile compounds were alcohols,
178
aldehydes and esters. In addition, the relative contents of 2-methylbutyl acetate, hexyl acetate, 2-hexenal, hexanal and
179
butyl acetate were higher in all volatile compounds, as shown by Lu et al. (2018). As shown in Table 4, the effects of
180
different drop shock conditions on variation of volatile compounds in apples were different. In general, aldehydes and
181
esters in samples shocked on marble were relatively higher than that of other contact material. This demonstrated that
182
high-intensitive drop shock stimulated the accumulation of aldehydes and esters. However, as for alcohols, the results
183
were various. The possible reason was that mechanical damage promoted fruit physiological metabolism, resulting in
184
the accumulation of aldehydes and esters. Up to now, there were few research results on the effects of mechanical
185
damage on the changes of volatile compounds in fruit.
186
3.5. Effect of Different Shock Conditions on Ethylene Biosynthetic Genes
187
As a plant gas hormone, ethylene is involved in the regulation of many important processes in plants. ACS and
188
ACO are two key enzymes for ethylene biosynthesis, which has significant meanings for the regulation of ripening and
189
senescence of fruits (Xu et al. 2016 a). As shown in Fig. 2, the gene expression levels of MdACS1 and MdACO1 in
190
apple fruit enhanced slowly when the shock height was 0-30 cm, and then weakened gradually with the increase of
191
shock height. This trend was consistent with the above results of ethylene production and respiratory rate. The
192
expression of MdACS1 and MdACO1 in samples shocked on marble at the height of 30 cm was over 10 fold higher than
193
that of untreated fruit. The other contact materials similarly enhanced the expression levels of ethylene biosynthetic
194
genes, although not as prominent as the effect of marble. Therefore, it was proved that reducing the impact in
195
transportation was of great significance for prolonging storage life and improving commercial quality of fruit in theory.
196
3.6. Effect of Different Shock Conditions on Ethylene Receptor Genes
197
Ethylene physiological action is finally completed through ethylene receptors and its signal transduction (Xu et al.
198
2016 b). As shown in Fig. 2, different shock treatment effectively improved the expression levels of ethylene receptor 5
199
genes in varying degree compared with the untreated apples. However, the enhanced effect of shock treatment of
200
marble on the expression of MdETR1 and MdCTR1 was the most obvious (p < 0.05) comparing to the other contact
201
materials. Furthermore, the effect of shock height on the expression of ethylene receptor gene was significant. Fig. 2
202
indicated that ethylene receptor gene expression reached the peak at the shock height of 30 cm, which was significant (p
203
< 0.05) compared with the control group. The possible reason for the decline in gene expression levels of ethylene
204
receptor with the increase of shock height was the repair and regulation of the fruit itself. The variation trend of
205
ethylene receptor gene expression was similar to the above results of ethylene biosynthetic genes, which indicated that
206
external damage seriously affected the ethylene signal transduction pathway of fruits. Thus it can be seen that
207
anti-shock package was necessary to reduce mechanical damage of fruits caused by vibration or shock.
208
4. Conclusions
209
Our present study investigated the influence of drop shock on physiological responses and ethylene biosynthetic
210
genes and ethylene receptor genes expression in harvested apple fruit. The results suggested drop shock dramatically
211
increased ethylene production, respiratory rate, soluble solid content, relative electrical conductivity, LOX activity,
212
MDA content and gene expression levels of MdACS1, MdACO1, MdETR1 and MdCTR1 in apple fruit. Furthermore,
213
firmness of harvested apples declined evidently after drop shock. High-intensitive drop shock stimulated the
214
accumulation of aldehydes and esters in apples. In conclusion, drop shock resulted in serious deterioration of fruit
215
quality and it is especially necessary to search a scientific and effective anti-shock measure. Further research has been
216
undertaken to examine changes of related proteins in fruits after drop shock with proteomics, which aims to look for the
217
underlying mechanisms of internal changes in fruits after drop shock.
218
Acknowledgements This work was supported by National Natural Science Foundation of China (31201434) and
219
Natural Science Foundation of Liaoning Province (20180550837).
220
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Table 1 Sequences of primers and probes for quantitative analysis of genes expression with real-time PCR. Name
Accession number
Product size
Sequence
MdACS1
U73815
146
MdACO1
DQ137850
129
MdETR1
DQ137848
118
MdCTR1
AY670703
159
18s rRNA
CV82635
87
5′TGAAATCTATGCAGCAACCGGCC3′ 3′CAACCCTAAATCCAGGGAAC5′ 5′ACCTTCCTTCCTCAAACAT3′ 3′CACCTCAATCTGGTCACCTAAG5′ 5′TGTTGGGAATGCTGTGAAGT3′ 3′ATGATTTGCTTGCAGCTTGG5′ 5′AACCTCCCATTGTTCATCGT3′ 3′CTTCTGGTGGTGCCATCCATTCA5′ 5′GGGTTCGATTCCGGAGAGG3′ 3′CCGTGTCAGGATTGGGTAAT5′
293 294 295 296 297 298
Table 2 Effects of different shock conditions on electrical conductivity, LOX activity and MDA content of apple fruit at 20 ± 2 ℃. Values are mean ± SE of triplicate samples. Drop height (cm)
Measuring item Shock treatment Relative electrical conductivity (%)
0
10
20
30
Marble
34.1±0.02Aa
46.5±0.15Ab
57.2±0.26Ac
71.5±0.35Ad
65.5±0.24
HDPE plastics
34.1±0.02Aa
40.2±0.13Bb
55.2±0.35Ac
65.1±0.48Bd
59.4±0.26
Corrugated board
34.1±0.02Aa
36.3±0.09Cb
42.5±0.54Bc
61.5±0.25Cd
55.1±0.05
Foamed plastics
34.1±0.02Aa
34.5±0.05Ca
45.0±0.15Bb
57.3±0.19Dc
51.2±0.02
90±0.25Aa
159±0.91Ab
164±0.36Ac
181±0.78Ad
179±0.51A
90±0.25Aa
140±0.84Bb
143±0.54Bb
160±0.59Bc
169±0.31B
90±0.25Aa
118±0.58Cb
125±0.57Cc
150±0.93Cd
148±0.57C
Foamed plastics
90±0.25Aa
99±0.72Db
110±0.86Dc
119±0.58Dd
115±0.84D
Marble
2.2±0.03Aa
4.2±0.05Ab
5.5±0.03Ac
5.8±0.28Ac
5.2±0.21A
2.2±0.03Aa
3.5±0.02Bb
4.5±0.05Bc
4.6±0.16Bc
4.1±0.06B
2.2±0.03Aa
3.1±0.16Bb
3.7±0.16Cc
4.2±0.24Bd
2.4±0.08C
2.2±0.03Aa
2.3±0.09Ca
2.4±0.49Da
3.4±0.38Cb
1.5±0.27D
Marble LOX activity HDPE plastics Corrugated board (min g–1)
MDA content HDPE plastics (nmol g–1) Corrugated board Foamed plastics 299
a
300
b
All means in the same column followed by different letters (A-C) are significantly (p <0.05) different. All means in the same row followed by different letters (a-d) are significantly (p <0.05) different.
301 302
Table 3 Standard sensor arrays and performance specification in electronic nose PEN 3. Number
Sensor
Performance
Notes
S1
W1C
Aromatic
Methylbenzene, 10 ppm
S2
W5S
Broadrange
NO2, 1 ppm
S3
W3C
Ammonium hydroxide
Benzene, 10 ppm
S4
W6S
Hydrogen
H2, 100 ppm
S5
W5C
Aromatic aliphatics
Dimethylmethane, 1 ppm
S6
W1S
Broad methane
CH3, 100 ppm 8
40
S7
W1W
Sulfur organic
H2S, 1 ppm
S8
W2S
Broad alcohol
CO, 100 ppm
S9
W2W
Sulfurchlorinate
H2S, 1 ppm
S10
W3S
Methane aliphatics
CH3, 100 ppm
303 304 305
Table 4 Effects of different shock conditions on variation of volatile compounds in apples.
9
306 307 308
Relative content (%)
unds
0 cm
ol
l
ate
anoate
tanoate
noate
309
50 cm
Marble
HDPE plastics
Corrugated board
Foamed plastics
Marble
HDPE plastics
Corrugated board
0.69 a
0.58 a
0.57 a
0.44 a
0.75 a
0.61 a
0.65 a
0.19 a
0.36 b
0.22 a
0.31 b
0.15 a
0.20 a
0.19 a
23.65 a
25.85 a
26.36 ab
29.65 b
19.63 c
20.58 ac
22.16 ac
1.54 a
1.37 a
1.29 a
1.21 a
1.93 a
1.77 a
1.62 a
-
-
-
0.02 a
-
-
0.01 a
24.16 a
22.57 a
21.69 a
20.60 a
26.96 a
24.48 a
23.96 a
0.29 ab
0.45 a
0.48 a
0.52 a
0.12 b
0.31 ab
0.28 ab
0.89 a
0.62 a
0.58 a
0.49 a
1.05 a
0.85 a
0.79 a
0.15 a
0.26 a
0.21 a
0.31 a
0.10 a
0.19 a
0.17 a
98.26 a
80.16 b
79.47 b
75.25 b
100.78 a
92.09 a
89.65 a
112.69 a
100.15 b
102.36 b
100.23 b
125.65 a
117.28 a
118.52 a
0.48 a
0.39 a
0.33 a
0.29 a
0.65 a
0.41 a
0.40 a
0.84 a
0.69 a
0.65 a
0.52 a
0.96 a
0.71 a
0.68 a
359.69 a
315.25 b
302.63 b
256.32 c
405.20 d
365.69 a
358.85 a
89.66 a
72.65 b
70.19 b
70.20 b
98.36 a
80.11 a
79.52 a
22.65 a
15.29 b
16.08 b
11.27 b
29.85 a
18.66 b
17.98 b
223.65 a
179.69 b
185.46 b
156.35 c
256.93 a
201.58 a
198.64 b
41.12 a
35.26 ab
34.98 ab
32.02 b
45.21 a
38.55 ab
37.89 ab
1.20 a
0.89 a
0.95 a
0.56 a
1.45 a
1.02 a
0.98 a
8.36 a
4.22 b
3.98 b
2.21 c
10.03 d
6.32 b
7.05 a
29.65 a
23.55 a
24.08 a
21.26 a
32.16 a
24.56 a
25.05 a
5.96 a
4.45 b
5.08 a
1.59 b
7.66 a
5.95 a
6.09 a
2.69 a
1.53 b
1.66 b
1.25 b
3.44 a
2.58 a
2.36 a
15.66 a
12.85 ab
13.09 a
10.36 b
17.56 c
14.26 a
13.18 a
1.27 a
0.96 a
0.85 a
0.63 a
2.59 b
1.58 a
1.67 a
8.21 a
7.52 a
6.36 ab
5.20 b
9.85 a
7.92 a
8.05 a
41.56 a
35.28 b
33.19 b
30.26 b
45.25 a
41.52 a
40.05 a
22.65 a
20.05 a
19.98 ab
18.24 b
25.23 a
22.69 a
21.59 ab
5.39 a
3.06 b
3.18 b
2.56 b
7.85 c
4.32 b
5.68 a
15.87 a
12.69 b
10.23 c
9.68 c
18.82 d
14.45 a
14.98 a
Declaration of interests
310 10
311 312
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
313 314 315
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
316
317 318 319 320 321 322
Highlights • Drop shock observably increased ethylene production and respiratory rate. • Drop shock enhanced LOX activity and MDA content . • Drop shock effectively improved gene expression levels.
323 324 325
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