Effect of Mechanical Damage on Organizational Structure of Postharvest Navel Orange Rind

DENG Lili, CUI Wenjing, YAO Shixiang, ZENG Kaifang,*

(1. College of Food Science, Southwest University, Chongqing 400715, China;2. Research Center of Food Storage & Logistics, Southwest University, Chongqing 400715, China)

Abstract: Mechanical damage is the main reason for the occurrence of oleocellosis in citrus fruit because it can cause the rupture of rind oil glands and the consequent symptoms of oleocellosis. Nevertheless, mechanical damage also favors the development of other rind disorders. More complicated physiological disorder symptoms appear in the orange rind after mechanical damage,which cannot be simply attributed to the development of oleocellosis. To better elucidate the initiation and progression of mechanical damage-induced orange rind disorders, the ultrastructural changes occurring in ‘Fengjie' navel oranges during these processes were investigated by transmission electron microscopy and scanning electron microscopy. The contents of pectin, cellulose and lignin, the rate of generation of superoxide anion radicals and lipoxygenase activity were also measured.Results indicated that after mechanical damage, the integrity of the cell membrane was lost and the cells of oil glands in the flavedo underwent an apparent degradation, resulting in the rupture of oil glands, and consequently the release of orange oil and the degradation of both the flavedo and albedo. During the early stage of storage after mechanical damage, the activity of lipoxygenase and the generation rate of superoxide anion increased steeply, and the content of protopectin reduced sharply. The contents of water-soluble pectin significantly increased from Day 7 to 11 relative to the control (P < 0.05). On the whole, the results from this study can provide some insights into the cause of the ultrastructural changes occurring in navel orange rind after mechanical damage.

Keywords: mechanical damage; ‘Fengjie' navel orange; cell ultrastructure; physiological rind disorder; oil gland

Physiological rind disorders reduce the shelf life and degrade the appearance of fresh citrus fruit. Most disorders do not affect the quality of the edible portion of the fruit[1-2].However, physiological rind disorders are important factors affecting external quality of citrus fruit for fresh consumption,and can cause massive commercial losses[3].

Oleocellosis, an important disorder of citrus, is caused by the action of phototoxic oils from oil glands within the rind,this disorder can occur at any time during harvesting, handling,storage, or marketing[4], and easily occurs in postharvest storage,especially when fruits are stored at room temperature[5-7]. It was reported that mechanical damage is the main reason for the occurrence of oleocellosis because it can cause the rupture of rind oil glands and the consequent upsurge of symptoms of oleocellosis[4]. Oleocellosis might result from several sorts of injuries including insect attack, hail incidence, wind,bruising, improper handling and via contact of damaged fruit with undamaged fruit[8]. Therefore, regarding to the effect of mechanical injuries on oil gland, mechanical damage has been widely applied in previous studies on the mechanism of oleocellosis in citrus fruits[6]. The histological and subcellular microstructural changes in healthy, oil-treated, and mechanically damaged fruits have been reported in several papers[9-12].

Nevertheless, mechanical injuries also favor the development of other rind disorders. For example, California navels that developed either postharvest pitting (PP) or stem-end rind breakdown (SERB) were both anatomically characterized by oil gland damage. However, both PP and SERB did not have apparent distinguishing anatomical traits,in which PP characterized by clusters of collapsed oil glands scattered over the surface of the fruit with the primary damage occurring in the cells enveloping oil glands[13].

Mechanical injuries are extremely common along the postharvest handling chain of citrus fruit. These injuries are defined as plastic deformations, superficial ruptures, or tissue damages caused by external forces[14]. Postharvest mechanical injuries, such as bruising, vibration damage during transportation and improper handling can increase the respiration rate and ethylene production of fruit[15-17], and destroy the membrane structure of fruit[18-19], thus affecting the microenvironment around fruit, while the happen of many rind disorders is directly connect with the relative humidity, the content of oxygen and carbon dioxide of the surrounding environment[20-22]. Moreover,the injury also promotes disruption of epidermal cells, triggering enzymatic reactions and, consequently, the appearance of darkened areas in the affected region due to the leakage of cell liquid and exposure to enzymatic action, which promotes the oxidation of phenolic compounds into quinones[23]. Therefore,disorder symptom appeared after mechanical damage on orange rind should be complicated, which cannot be simply attributed to the development of oleocellosis. As demonstrated from previous researches, the organizational structure changes in oleocellosis-infected citrus rind have been widely studied, therefore, to well distinguish the difference between oleocellosis and the disorder caused by mechanical damage, the understanding of the organizational structure of postharvest citrus rind after mechanical damage is of great importance.

In this paper, in order to well illuminate the disorder symptom of citrus rind after mechanical damage, the systemic and detailed changes of the cellular ultrastructure and the contents of structural materials in ‘Fengjie' navel orange rind treated by mechanical damage were investigated to provide some basic information for the better understanding and distinguish those physiological rind disorders caused by mechanical damage.

1 Materials and methods

1.1 Materials and reagents

Citrus fruits (Citrus sinensis L. Osbeck cv. Fengjie) were harvested early in the morning from the Beibei District of Chongqing City, China and then transported immediately to the laboratory. All fruits were uniform in size and color and had no physical injuries or infections on the rind.

Phosphate buffer, hydroxylamine hydrochloride, sulfanilic acid, α-naphthylamine, sodium soyate, ethanol, vitriol, carbazole,D-(+)-galactuonicacid, hexane, acetobrom, tert-butyl alcohol,NaOH, glacial acetic acid, anthranone, glutaraldehyde, isoamyl acetate, glutaraldehyde, osmium tetroxide Chongqing Beibei Chemical Reagent Plant.

1.2 Instrument and equipment

HZQ-F100 oscillating cultivating box Taicang Experimental Device Factory; Tecnal-10 transmission electron microscope (TEM) Philips Co. Ltd.; UV-1000 ultravioletvisible spectrophotometer Techcomp (China) Co. Ltd.;3400N scanning electron microscope (SEM) Hitachi Co.Ltd.; DZF6032 vacuum drying oven Keelrein Inc.; D7200 digital camera Nikon Inc..

1.3 Methods

1.3.1 Mechanical damage and sampling

Citrus fruits were vibrated continuously at 100 r/min for 72 h, citrus fruits without any treatment were used as control fruit. Then every fruit of these two groups were individually packaged in plastic polyethylene bags (150 mm × 150 mm,0.015 mm) and sealed and held at 5 ℃ and relative humidity of 85% - 90% for 11 d. A total of 24 fruits were used in each treatment, and each trial was performed in three replicates. The mechanically damaged part (the browning section) of the rind from each fruit was collected for further study, a part of these rind were combined, cut into small pieces, frozen immediately in liquid nitrogen, and stored at -80 ℃ prior to use, and another part of these rind were collected directly for TEM and SEM analysis.

Pictures of the mechanically damaged part were taken using a digital camera.

1.3.2 Measurements of rind collapse index and discolouration index

Rind collapse and discolouration index were assessed using a subjective scale according to the methods of Knight et al[23]: 0 (nil), 1 (very slight), 2 (slight), 3 (medium) and 4 (high).The score was computed as follows.

1.3.3 Measurements of LOX activity and generation rate of superoxide anion radicals

Activity of lipoxygenase (LOX) were determined according to the method of Chen Kunsong et al[24]. One unit of LOX activity equaled a change of 0.01 in absorbance per minute, as measured at 234 nm (U). The generation rate of superoxide anion radicals were determined according to the method of Wang Aiguo et al[25].

1.3.4 Measurements of pectin, cellulose and lignin contents

Water soluble pectin and protopectin contents were determined by conducting carbazole-vitriol colorimetry[26].Cellulose contents were determined according to the method of Niu Sen[27]. Lignin contents were determined according to the method of Deng Lili et al[28]and was represented as the absorbance at 280 nm per 1 g rind (OD280nm).

1.3.5 SEM measurement

Samples for SEM were prepared according to the method by Agustí et al[4]with slight modifications. Rind pieces (2 mm × 2 mm × 1 mm) from the exposed face of the fruit were vacuum-infiltrated for 10 min in 2.5%(V/V) glutaraldehyde in 50 mmol/L phosphate buffer (pH 7.2)before the samples were fi xed in the same solution for 2 h at 4 ℃.The material was then rinsed several times with the same buffer, dehydrated respectively in a graded ethanol series(50%, 60%, 70%, 80%, and 90%) for approximately 5-7 min,and dehydrated with 100% ethanol twice for approximately 7 min. Subsequently, the material was further dehydrated in a graded tert-butyl alcohol series (50%, 70%, 80%, 90%,and 95%) for approximately 7 min and dehydrated with 100%tert-butyl alcohol twice for approximately 7 min. All rind pieces were fixed and dehydrated as described above. The material was critical-point dried in liquid carbon dioxide and mounted on SEM stubs. The mounted material was coated with gold in a E-6100 sputter coater, performed on a S-3400N SEM at an acceleration voltage of 10-15 kV.

1.3.6 TEM measurement

Samples for TEM were prepared according to the method of Bennici et al[12]. Rind pieces (1 mm × 1 mm ×1 mm) from the exposed face of the fruit were vacuuminfiltrated for 10 min in 2.5% glutaraldehyde in 50 mmol/L phosphate buffer (pH 7.2) and fixed in the same solution overnight at 4 ℃. Subsequently, the pieces were rinsed with the same phosphate buffer thrice, post-fixed in 1%osmium tetroxide for approximately 2 h, dehydrated with an ethanol series (50%, 60%, 70%, 80%, 90%, and 100%), and embedded in LR white resin. Ultra-thin sections (60-80 nm)were collected on collodion-coated copper grids, stained with uranyl acetate and lead citrate, and viewed under a Tecnal-10 TEM.

1.4 Statistical analysis

All the statistical analyses for this experiment were performed using SPSS 17.0 software. Data were expressed as mean ± SD.Data were analyzed using Student's t-test. Differences at P< 0.05 were considered significant. All experiments were replicated thrice. The fi gures were drawn using the Excel 2016 software.

2 Results and Analyses

2.1 Effects of postharvest mechanical damage on the rind appearance of navel orange rind

Mechanical damage had a serious effect on the rind appearance of navel oranges. As shown in Fig. 1A, no collapse and no discoloration were found in untreated rinds. The surface of healthy citrus fruits had a uniform color and the absence of spots. After Day 1 of the induction of mechanical damage (Fig.1B), the oil glands were depressed. Meanwhile, the color of the region turned brown. After 7 d (Fig. 1C), the lesion of disorder was gradually enlarged, and the rind had a reddish-brown color.

Fig. 1 Effect of postharvest mechanical damage on appearance of navel orange rind during storage

2.2 Effects of postharvest mechanical damage on collapse and discolouration index of navel orange rind

Fig. 2 Effect of postharvest mechanical damage on collapse index(A) and discolouration index (B) of navel orange rind during storage

As shown in Fig. 2, both the collapse and discolouration index increased slowly in the control fruits, whereas these scores in the mechanically damaged fruits increased sharply with storage time (P < 0.05), especially in the fi rst 3 days of storage.These results indicate that mechanical damage had a serious effect on the appearance of navel orange rind. By the 3 d of storage, the collapse and discolouration index of mechanical damaged fruit were 2.81 times and 1.77 times compared to control fruit (P < 0.05).

2.3 Effects of postharvest mechanical damage on LOX activity and generation rate of superoxide anion radicals of navel orange rind

Fig. 3 Effect of postharvest mechanical damage on lipoxygenase activity (A) and generation rate of superoxide anion radicals (B) in navel orange rind during storage

As shown in Fig. 3A, the LOX activity increased to peak and then decreased. Postharvest mechanical damage could delay the peak time of LOX activity in navel orange rinds, the LOX activity of control group reached peak values on Day 1,and then decreased to a relative lower level, while the LOX activity of mechanical damaged fruit reached to peak on Day 3 (P＜0.05), approximately 1.64 times of the control fruit and kept at a relative higher level. As shown in Fig. 3B, superoxide anion generation rate reached to peak at Day 3 in all groups and then decreased gradually. The generation rate of superoxide anion radicals in mechanical damaged fruit were 1.6, 1.6 and 1.3 times higher than those in the control fruits on Day 1, 5 and 7 d after treatment, respectively (P < 0.05).

2.4 Effects of postharvest mechanical damage on pectin,cellulose and lignin contents of navel orange rind

As shown in Fig. 4A, the protopectin content decreased in all groups during storage. The protopectin content of mechanical damaged fruit sharply decreased at the first 5 d.The protopectin contents in control fruit were 1.3, 2.1 and 1.6 times higher than those in the mechanical damaged fruits on 1, 3 and 5 d after treatment, respectively (P < 0.05). As shown in Fig. 4B, the water-soluble pectin content increased and then decreased gradually in all groups during storage. The water-soluble pectin content of mechanical damaged fruit sharply increased to a higher level at later time of storage.The water-soluble pectin contents in mechanical damaged fruit were 1.3, 1.4 and 2.1 times higher than those in control fruits on 7, 9 and 11 d after treatment, respectively(P < 0.05). As shown in Fig. 4C, the cellulose content increased and then decreased in control group during storage, and a sharply increase were found in mechanical damaged fruit. The peak value of cellulose content of mechanical damaged fruit was approximately 1.61 times of the peak value in the control group. As shown in Fig. 4D, the lignin content increased and then decreased in control group during storage, and the lignin content of mechanical damaged fruit were lower than control fruit during the whole storage.

Fig. 4 Effect of postharvest mechanical damage on protopectin (A),water-soluble pectin (B), cellulose (C) and lignin (D) contents of navel orange rind during storage

2.5 Effects of postharvest mechanical damage on the oil glands, flavedo and albedo of navel orange rind

Mechanical damage imposed a serious effect on the oil glands and the surrounding tissues. As illustrated in Fig. 5A1,round or oval-shaped oil glands in healthy tissue were located at the flavedo of citrus fruit rind, which is the main location of orange oil, and the oil glands were composed of thousands of cells that were arranged in an orderly layered structure.After the first day of the induction of mechanical damage(Fig. 5A2), the oil glands showed severe depressions, such that whole oil glands were pitted and shrunk. The orderly arranged cell layers of the oil gland had disappeared, the boundary cell layers, not the inner cells, were initially degraded; faults and a black cavity could be observed in the center of the oil glands by 7 days of storage, which showed that orange oil was released from the oil glands (Fig. 5A3).Mechanical damage caused the rupture of the epidermis above the said oil glands; and the openings produced by mechanical damage acted as a route for surface oil movement into the cortex, thereby leading to rapid cortical degeneration.

The citrus rind or pericarp is composed of two tissue layers: the flavedo and the albedo. The flavedo is the tissue in which the oil glands are embedded, which comprised of the epicarp, hypoderm, and outer mesocarp. The flavedo cell layers are always arranged in an orderly and compact manner; the interspaces of the cells appeared relatively small in the healthy rind tissue (Fig. 5B1). Compared with the albedo, the flavedo is more sensitive to mechanical damage. After 1 d of mechanical damage (Fig. 5B2), the flavedo lost the orderly arranged layered structure, some larger cell interspaces could be observed, and some fragments were present in the flavedo, these results imply that the cell layers of the flavedo were degraded. By Day 7 of induction (Fig. 5B3), the damaged areas were easily detected by their flattened and collapsed appearance while the intercellular space gradually expanded.

Fig. 5 Effect of postharvest mechanical damage on ultrastructure of oil gland (A) (100 ×), flavedo (B) (500 ×) and albedo (C)

The inner mesocarp, or albedo, is commonly referred to the colorless inner spongy tissue. As shown in Fig. 5C1,intercellular air spaces were present between the cells, thereby showing a typical spongy morphology of albedo tissue. No signs of cell disruption or collapse were observed; the cells were well compacted and packed in healthy albedo. During early storage stage (Fig. 5C2), some intercellular air spaces and hollow organization in albedo tissue could be found. With prolonged storage time, the number and size of the intercellular air spaces were increased and the albedo became more loose (Fig. 5C3).

2.6 Effects of postharvest mechanical damage on the cell shape and cell wall of navel orange rind

Major structural changes were observed in the overall profile of the rind after mechanical damage. As illustrated in Fig. 6A1, healthy rind sample tissue was well preserved based on TEM, and all cells had intact organelles and membrane systems. The cells were oval in profile and enveloped by intact membranes, with several randomly distributed organelles in the cytoplasm. Marked differences were noted in the ultrastructure of cells from healthy areas or from the mechanically damaged rind of fruits. Ultrastructural degradation was already apparent in treated fruits. Mechanical damage had serious effects on the cell shape (Fig. 6A2, A3). By the Day 7 of storage, whole cells had completely lost their original shapes, some mechanically damaged cells contained a collapsed protoplast, in which few structures could be indentified.

Fig. 6 Effect of postharvest mechanical damage on cell shape (A)and cell wall (B) of navel orange rind during storage

Cell wall acts as one of the physical defensive barriers of plant. This structure is composed of a primary wall, a secondary wall, and the middle lamella. The middle lamella,sometimes called the pectin layer, is the outer wall of the cell that is rich in pectin, this structure is the cementing layer of the cell. As shown in Fig. 6B1, the cell wall of healthy rind tissue under TEM was composed of densely packed fibrils in an electron-translucent matrix. The middle lamella was visible as a more electron-dense region between the cell walls of adjacent cells, which took on a white-black-white strip;the black strip represented a high electron density. After 1 d(Fig. 6B2), the cell wall of mechanically damaged fruits began to appear irregular and the cell walls were bent; the cells presented a collapsed cell shape. However, by Day 7(Fig. 6B3), cell collapse and extreme degradation had occurred.The cell wall had been degraded to fi laments or strips as a result of losing the turgor pressure of the cell.

2.7 Effects of postharvest mechanical damage on the chloroplasts, mitochondria and nuclei of navel orange rind

In healthy tissue, chromoplasts were generally round to oval shaped and exhibited an intact outer membrane, a folder inner membrane, and lightly stained plastoglobuli. Mature chloroplasts contained numerous white starch particles and plastoglobuli (Fig. 7A1). After 1 d of treatment (Fig. 7A2),the chloroplasts appeared swollen and deformed, whereas the thylakoids were unstacked and randomly arranged. The chloroplasts showed signs of distorted thylakoid membranes,as well as reduced size and number of starch granules. By Day 7 (Fig. 7A3), no chloroplasts were detected in rind cells, and the lamellar structure had been completely degraded into scattered fragments; only the increased number of osmiophilic globules remained.

In healthy rind cells, mitochondria maintained their“double membrane” structure, with apparent internal cristae(Fig. 7B1). However, after 1 d of treatment (Fig. 7B2), the mitochondria were flattened to an oval shape, the membrane systems showed signs of degeneration, and the cristae were unclear or had even disappeared. Moreover, the number of mitochondria was reduced. By Day 7 (Fig. 7B3), no organelles could be found in the cells in mechanically damaged fruits,which meant that the energy capacity was reduced and cell aging had advanced.

Fig. 7 Effect of postharvest mechanical damage on chloroplasts (A),mitochondria (B) and nuclei (C) of navel orange rind during storage

In healthy cells, the nucleus is generally oval shape and enclosed by a double nuclear membrane. An oval-shaped and dark-colored nucleolus is located at the center of the nucleus(Fig. 7C1). After 1 d of mechanical damage (Fig. 7C2), the nucleus was partially deformed and resembled a tadpole, and the nucleolus had swollen. The membrane around the nucleolus had widened or even ruptured, and the nucleus was opaque and nonhomogeneous, while as mentioned above, by Day 7, no organelles were left (Fig. 7C3).

3 Discussion

Our results indicated that mechanical damage had a serious effect on the rind appearance of navel oranges. The oil glands were depressed and the color of the region turned brown, and then a reddish-brown color appeared in damaged region. These symptoms were accordance with the results of the collapse and discolouration index.

Oil gland were wrapped by several layers of thick boundary cells[6], and in citrus fruit, the oil glands span a region from the flavedo of the fruit to the depth of the albedo[29]. As visualized by SEM, the cells of oil gland were degraded in the flavedo of citrus fruits under the inducement of mechanical damage. Conversely, the degraded flavedo causes the rupture of oil glands and the release of orange oil.Previous studies demonstrated that orange oil can promote rind tissue degradation[30]. Once endogenous orange oil entered the citrus rind tissue, transition metal ions, such as Fe and Cu,could catalyze the formation of phenolic hydroxyl, increasing the available ROS in orange rind. ROS rapidly attacks the cell membrane and cell wall of the pericarp. Besides, ROS could be toxic to cellular organelles, which agrees with the reduced organization of the oil glands and the browning at the lesions[31-32].

The cell wall is one of the physical defensive barriers of plant cells. Our fi ndings indicated that cell walls were gradually degraded during storage; cell walls underwent slight distortion before they were fi nally degraded into fragments, in accordance with the results of Maia et al[17]. Knight et al[5]speculated that cell wall thickening and collapse are most likely to be attributed to swelling and folding, respectively, of the cell walls. These results suggested that mechanical damage could accelerate cell wall breakdown because of losing cell turgor pressure.

Mechanical damage can also cause the degradation of the cortex cells in citrus fruit rind. Our results indicated that mechanical damage could cause the loss of membrane integrity.The degradation of the membrane system was the earliest sign of cell damage; thus, the discontinuity of the membrane systems can be used as an indicator of cell senescence and destruction[33]. The destruction of the membrane system may lead to the loss of cellular osmoregulatory capacity and cellular liquids. In addition, after the degradation of the membrane system, the regionalization of separate cell systems was destroyed; consequently, organelles could be destroyed without the protection of membrane systems, including the cell wall,tonoplast, mitochondria, and chloroplast.

Cells of mechanically damaged fruit lose their intact membrane systems, subsequently, the structure and function of organelles are partially lost, including those of the chloroplast, mitochondria, and tonoplast, among others. Some ultrastructural changes of these organelles were visualized by TEM in this paper; the disappearance of chloroplast thylakoids and mitochondria cristae, as well as the destruction of the tonoplast, was observed. At the end of storage, few of the organelles can be found in mechanically damaged fruits; the few chloroplasts that were found had serious degradation.

Chloroplast is the site of ROS generation, this organelle is the most vulnerable to ROS attack[34]. The degradation of chloroplasts is bound to affect the chlorophyll synthesis,thereby leading to the color change of citrus rind lesions.In the present study, we found that starch particles completely disappeared in severely degraded chloroplast,as well as the emergence of a large number of denser grana in the mechanically damaged fruits. Shomer et al[8]found that immature citrus affected by oleocellosis show some abnormal behavior in the rind color changes because of the formation of giant chloroplasts that contain denser grana. The chloroplast degradation and destruction of the regionalization system explained the brown or light green rind color of oleocellosis-affected citrus fruits[33]. Once the regionalization system of a cell is broken, the browning reaction of polyphenol in tonoplasts and enzymes (PPO, POD, etc.) in the cytoplasm is initiated[35]. This change is consistent with the increase of the discolouration index.

Cellulose, pectin and lignin are the main macromolecules that form the main constituent of the cell wall in most plants. The degradation of these macromolecules, especially protopectin, could cause the loss of the connection among cells and the disintegration of the cells. Our results indicated that the mechanical damage promote the conversion of protopectin to water-soluble pectin. Besides, the increase of lipoxygenase activity and content of superoxide anion were consistent with the ultrastructural changes mentioned above.

4 Conclusion

In conclusion, as a widely used method in studies on the mechanism of oleocellosis in citrus fruits, mechanical damage had a significant effect on the oil glands and the ultrastructure of rind tissue The disorder symptom that appeared after mechanical damage on orange rind disorder should be more complicated, which cannot be simply attributed to the development of oleocellosis.