1‐Methylcyclopropene (1‐MCP) retards the senescence of Pteridium aquilinum var. latiusculum by regulating the cellular energy status and membrane lipid metabolism

Abstract 1‐MCP is an ethylene inhibitor which can delay the ripening and senescence of fruits and vegetables effectively. Pteridium aquilinum var. Latiusculum (PA) is one of the wild vegetables which is famous and nutrient in China. However, the mechanism of PA preservation treated with 1‐MCP has not been reported. Consequently, the effects of postharvest 1‐MCP treatment on the changes in quality, energy metabolism, and membrane lipid metabolism of PA were investigated in this study. The results indicated that 1‐MCP treatment could effectively inhibit the decreases in firmness, titratable acid (TA) content and the increases in weight loss rate, malondialdehyde (MDA) content, membrane permeability, and membrane lipid metabolism‐related enzymes in PA. The cellular energy charge (EC) and the levels of ATP, ATP/ADP, and ATP/AMP, the activities of energy metabolism‐related enzymes, NAD+, and NADH were maintained, and the decreases in unsaturated fatty acids and the ratio of unsaturated‐to‐saturated fatty acids in the membrane of PA cells were effectively retarded by 1‐MCP treatment. A positive correlation was observed between cellular ATP levels and the ratio of unsaturated‐to‐saturated fatty acids, while negative correlations were observed between the ratio of unsaturated‐to‐saturated fatty acids and both lipid peroxidation and membrane permeability. These results indicated that higher levels of energy status, unsaturated‐to‐saturated fatty acid ratios, and lipid metabolism in the membrane could preserve the membrane integrity of postharvest PA and effectively extend its shelf life.

popular in both domestic and international markets. It has become the leading variety of wild vegetables for export in Northeast China, creating great economic value (Hao et al., 2021). Nevertheless, PA usually grows deep in the mountains and woodlands that are far from the sales places. Ripening and senescence during transport cause considerable losses not only for PA but also for other wild vegetables.
Fruits and vegetables are still living organisms carrying out a series of living activities after harvest. Ripening and senescence processes easily occur due to the transient harvest period of PA during spring in Northeast China, which creates difficulties for its storage and preservation. Previous studies indicated that the energy supply of fruit and vegetable cells affects a series of physiological metabolic possesses during postharvest ripening and that postharvest quality changes were closely related to the energy dissipation of fruit and vegetable cells (Aghdam et al., 2018;Jin et al., 2014;Qu et al., 2006;Saquet et al., 2000;Wang et al., 2013). Adenosine triphosphate (ATP) is the primary energy resource found in cells. It plays an important role in maintaining the membrane integrity and energy status change of postharvest fruit and vegetable cells during the ripening process (Saquet et al., 2000(Saquet et al., , 2003. Some previous studies have also found that membrane integrity affects the browning of fresh-cut pears and litchi fruit (Jiang et al., 2018;Li et al., 2017). Therefore, changes in the energy metabolism within cells could affect the quality and decay of postharvest crops by impacting the integrity of cell membranes.

1-Methylcyclopropene (1-MCP) is an effective preservative for
fruits and vegetables since the ripening and senescence of fruits and vegetables will be inhibited. 1-MCP has the advantages of high efficiency, safety, nontoxicity, and no residue. It is currently widely used in postharvest horticultural products. There have been sufficient studies to show that 1-MCP treatment can extend shelf life efficiently (Kou et al., 2020;Kubo et al., 2003;Luo, 2007;Mata et al., 2018;Rocculi et al., 2009;Sharma et al., 2010;Sivakumar et al., 2010;Zhu et al., 2013).
Although 1-MCP has been widely used to improve the quality of postharvest vegetables, no study has focused on changes in the energy status, membrane fatty acid content, and membrane lipid metabolism of PA after 1-MCP treatment. Therefore, the objective of this study was to explore the relationship between the changes in energy status, membrane fatty acids, membrane lipid metabolism, and quality ripening of PA after 1-MCP treatment.

| Experimental materials and treatments
All the PA materials for the experiment were harvested from Heilongjiang Beian Agricultural Reclamation Tianyunshan Products Co., Ltd of Heihe, within Heilongjiang Province, China. All the PA materials were harvested on May 15th, 2020. The collection was based on their uniformity of size and maturity with the absence of diseases and mechanical damage. The length of each PA was between 20 and 25 cm. The whole harvesting process followed the local standards of Heilongjiang Province (Technical regulation for harvesting and postharvest treatment of Pteridium aquilinum var. latiusculum). The samples were quickly transported to the College of Food Science, Northeast Agricultural University after harvesting.
PA samples were rinsed using distilled water to remove dirt and dust on the surface. After air drying, 400 samples were evenly divided into the treatment group and control group. All the samples in the treatment group were fumigated using a 1 μL/L 1-MCP solution for 24 hr in an incubator (RGX-80B, Shanghai Kuntian Laboratory Instrument Co., Ltd) without controlled atmosphere according to the results of our previous experiment. Others in the control group were fumigated by distilled water. After fumigation, all the samples in both groups were transferred to 4℃ and 90%-95% relative humidity (RH) conditions. High RH is arranged to reduce the effect on energy level and membrane integrity of water loss. All the determinations were performed every three days, and each treatment contained three biological repeats to guarantee the accuracy and reliability of the determination.

| Determination of firmness, titratable acidity, weight loss rate, and ethylene production
The PA firmness was measured using a texture analyzer with a probe with a diameter of 2 mm (TA-XTplus2, Stable Micro Systems Ltd, UK).
The puncture speed and puncture distance of the texture analyzer were 2 mm/s and 12 mm, respectively. Ten samples were selected randomly from each treatment to calculate their average. Each sample was straightened before determination. The firmness was measured at the middle point of each PA sample length. Firmness was expressed as N.
Randomly selected PA tissue (10 g) from each group was used to measure the titratable acid (TA) content of PA. Samples were put into a mortar to be homogenized by continuous grinding. Then, all the residue was transferred to a 100 ml volumetric flask and filtered 30 min later. A mixture of 20 ml filtrate and two drops of 1% phenolphthalein indicator was titrated by 0.1 M sodium hydroxide.
Titration was not considered complete until the solution appeared pink and did not fade within 0.5 min (pH=8.1-8.3). The amount of sodium hydroxide solution was recorded, and this step was repeated three times to guarantee accuracy. Finally, titration of distilled water instead of filtrate was used as a blank control. The TA content was calculated by the following equation: V represents the total volume of the sample extraction solution (mL); Vs represents the filtrate volume (mL); c represents the concentration of sodium hydroxide solution (mol/L); V 1 represents the volume of sodium hydroxide solution needed to titrate the filtrate (mL); Vs × m V 0 represents the volume of sodium hydroxide solution needed to titrate distilled water (mL); and m represents the sample quality (g).
The weight loss rate of PA in both two groups was calculated according to the following equation: m 0 represents the original weight of PA sample before the storage; m i represents the weight of each determination during the storage.
The determination of ethylene production was measured as following steps: 15-20 PAs were randomly chosen in each group and placed into sealed glasses, respectively. 1 ml gas from the head-space of the container was taken and measured using a gas chromatographer (Agilent 7890B). The temperatures of the oven, gasifier, and FID were at 90℃, 140℃, and 200℃, respectively. Carrier flow rates of nitrogen, hydrogen, and air were 30, 30, and 300 ml/min, respectively.

| Determination of lipid peroxidation and membrane permeability
Malondialdehyde (MDA) is one of the main products generated from membrane lipid peroxidation. The MDA content is usually regarded as an index that reflects the extent of membrane lipid peroxidation.
The reaction of MDA with proteins and nucleic acids changed the configuration of these macromolecules and resulted in a loss of their biological functions. Therefore, the accumulation of MDA could cause damage to the membrane and organelles to a certain extent.
A mixture of 1.0 g of PA sample tissue and 5.0 ml of 100 g/L trichloroacetic acid (TCA) solution was ground. The supernatant was extracted from the homogenate by centrifugation at 4℃ and 10,000 × g for 20 min. Then, 2.0 ml of 0.67% thiobarbituric acid (TBA) was added to 2.0 ml of supernatant. After mixing, the mixture was boiled in a boiling water bath for 20 min. Then, it was quickly cooled in an ice bath and centrifuged one more time (4℃, 10,000 × g for 20 min). The absorbance of the supernatant was recorded at 450, 532, and 600 nm. The MDA concentration of the sample extract could be calculated by means of the following equation: Then, the MDA content of the sample extract could be calculated according to the following equation. The results were expressed as μmol/g on a fresh weight (FW) basis: c represents the MDA content of the reaction mixture (mmol/ kg); V represents the total volume of the sample extract (mL); Vs represents the total volume of the sample extract required for determination (mL); and m represents the sample weight (g).
The membrane permeability of PA was measured using the relative leakage rate (RLR). The sample tissue disks were removed using a hole punch from the equatorial region of each PA. Ten disks from each of the samples from each treatment were immersed in 20 ml of deionized water at room temperature for 30 min and then vibrated for 10 min. The samples were removed, and the remaining water on the surface was dried using filter paper. Then, the samples were put into a boiling tube and immersed in 20 ml of deionized water again.
The initial electrical conductivity of the solution was measured using a conductivity meter at a constant temperature of 20℃-25℃, and it was represented by γ 1 . Then, the solution with disks was moved into a boiling water bath for 15 min and allowed to cool, and the final electrical conductivity of the solution was measured at a constant temperature of 20℃-25℃. The final electrical conductivity of the solution was represented by γ 0 . The relative electrical conductivity was represented by γ e and was calculated by the following equation:

| HPLC analysis of ATP, ADP, and AMP
The extraction and determination of ATP, ADP, and AMP were ex- Elution was executed using a linear gradient program with 75% to 100% A and 0% to 25% B for 7 min. The temperature of the column oven was 25℃. The flow rate was 1.0 ml/min. 15 μL of each sample was injected into the HPLC system for the analysis of ATP, ADP, and AMP concentrations according to the external standard curve. The energy charge was calculated by the following equation: The results were expressed in μg/kg.

| Determination and analysis of membrane fatty acids
The membrane lipids were extracted and quantified according to water were added to the solution sequentially. 8 ml of a 2% NaOHmethanol solution was added to the extraction and then connected to a reflux condenser. Reflux was performed in an 80℃ water bath until the oil disappeared. 7 ml of 15% boron trifluoride-methanol solution was added from the top of the reflux condenser and refluxed in an 80℃ water bath for 2 min. Then, the flask was removed from the water bath and cooled to room temperature immediately. Next, 10-30 ml of n-heptane was added to the solution and vibrated for 2 min. After vibration, a saturated NaCl solution was added, and the solution was separated into different layers. 5 ml of from the top of the n-heptane extraction solution was transferred to a test tube, and then 5 g of Na 2 SO 4 was added with vibration for 1 min. After standing for 5 min, the solution in the upper layer was used to test the content of fatty acids.

| Activities of energy metabolismrelated enzymes
The activities of succinate dehydrogenase (SDH), cytochrome c oxidase (CCO) H + -ATPase, and Ca 2+ -ATPase were determined using enzyme-linked immunosorbent assay (Elisa) kits (Shanghai Jingkang Bioengineering Co., Ltd). The operation was carried out according to the instructions and information provided by the manufacturer.

| Determination of NAD + and NADH contents
The activities of NAD + and NADH were determined using enzyme-linked immunosorbent assay (Elisa) kits (Shanghai Jingkang Bioengineering Co., Ltd), respectively. The operation was carried out according to the instructions and information provided by the manufacturer. The activities of NAD + and NADH were expressed as ng/gprot.

| Activities of membrane metabolismrelated enzymes
The determination of lipoxygenase (LOX) activity, lipase (LPS) activity, and phospholipase (PLD) activity was using lipoxygenase assay kit, lipase assay kit, and phospholipase assay kit (Shanghai Jingkang Bioengineering Co., Ltd), respectively. Their operation was carried out according to the instructions and information provided by the manufacturer. The activities of LOX, LPS, and PLD were expressed as ng/gprot.

| Statistical analysis
The experiment was executed under a completely randomized design. All the data from this experiment were calculated using Microsoft Excel 2016 and plotted using Origin Pro 8.5. The data were presented as the means ±standard deviation (SD) and their significant difference was analyzed by paired-sample t test in Origin.
p < .05 was regarded at a significant level according to the test.
Correlation coefficients of intracellular indexes and membrane indexes were calculated using Origin Pro 8.5. The correlation was at a significant level when p < .05 (Table 1).

| Firmness, TA, weight loss rate, and ethylene production of PA
Firmness is usually regarded as an important index to evaluate the appearance quality of vegetables. The firmness of PA in both the control and treatment decreased gradually during the whole experiment. The decreasing trend of 1-MCP treatment was obviously delayed compared with that of the control group, and the firmness gap between the two groups tended to be larger as time went on. In contrast, 1-MCP effectively maintained PA firmness at a relatively high level during storage. This result indicated that 1-MCP could be an effective preservative to prolong the shelf life of PA during storage.
TA is an important component of vegetable quality and an important factor affecting its flavor quality. The TA content in both the control and treatment groups decreased gradually during storage.
Samples treated with 1-MCP exhibited a slower decrease in TA content than the control. However, the TA content of samples treated Weight loss rate is an essential index that evaluating the quality of vegetables. Water loss was the main reason for the weight loss in PA. As shown in Figure 1c, the weight loss rates of PA in both groups increased gradually during the storage. The increasing trend of 1-MCP treatment was significantly lower than that of the control.
It indicated that 1-MCP treatment could inhibit the weight loss of PA and maintain it at a high level effectively.
As shown in Figure 1d, the ethylene production of PA in both groups increased gradually during the whole storage time. However, the endogenous ethylene production of 1-MCP treated was obviously lower than that of the control during the same storage time.
1-MCP has been demonstrated to be an effective ethylene inhibitor which could delay the ripening and senescence in the preservation of fruits and vegetables (Rocculi et al., 2009;Salvador et al., 2004;Sharma et al., 2010;Sivakumar & Korsten, 2010

| Membrane permeability and lipid peroxidation
The relative leakage rate of both the control and treatment increased gradually during storage. Compared to that of the control, Therefore, 1-MCP treatment could not only maintain the firmness and TA of PA, but also inhibit the damage to the membrane effectively according to the results of this study.

| Energy status
Many previous studies have proven that cellular energy supply is an important factor in controlling ripening and senescence in plants after harvest (Azad et al., 2008;Jiang et al., 2007;Wang et al., 2013).
Therefore, inadequate energy supply or a decrease in cellular energy TA B L E 1 Correlation coefficients of intracellular indexes (energy charge, ATP, ATP/ADP, and ATP/AMP) and membrane indexes (lipid peroxidation, membrane permeability, and ratios of unsaturated-to-saturated fatty acids) in Pteridium aquilinum var. latiusculum during the storage. Correlation coefficients were calculated using Spearman's Correlation in Origin Pro 8.5. p < .05 indicated that a significant correlation was observed between the indexes could affect the changes in fruit quality during storage and could even negatively affect physiological disorders and pathogen infection (Huang et al., 2019). In this study, the ATP content showed an Once the senescence process was triggered, the ATP content started to decrease. To maintain the homeostasis of the cellular energy level during storage, the ADP and AMP contents were increased. After increasing for a few days, the ADP and AMP contents also decreased rapidly (Figures 2-4).
The energy charge was calculated to reveal the overall energy status of the cells in different stages (Jiang et al., 2007;Pradet & Raymond, 1983). ATP/ADP is an important factor related to mitochondrial function by enhancing or inhibiting glycolysis metabolism (Maldonado & Lemasters, 2014). ATP/ADP and ATP/AMP not only are related to senescence, but also affect the incidence of physiological disorders and the alteration of cellular macromolecules F I G U R E 1 Changes in PA firmness (a), TA content (b), weight loss rate (c), and ethylene production (d) in both the control and the 1-MCPtreated sample during postharvest ripening. Each data point is the mean ±standard deviation (SD) of three replicates. Asterisks at each time point represent significant differences according to t test at p < .05 (Wang et al., 2003). In this study, the energy charge, ATP/ADP and ATP/AMP all increased in the early storage period and then de- permeability and damage to membrane integrity. These changes were due to the limited availability of both ATP content and energy charge level (Jiang et al., 2007;Saquet et al., 2003). Finally, a significant negative correlation was observed between ATP content and membrane permeability in the harvested PA tissues. Therefore, the senescence process of harvested PA might be closely related to the cellular energy status. Maintaining the ATP level and cellular energy status could retard the quality deterioration of PA.

| Changes in membrane fatty acid contents
Changes in membrane fatty acid constituents are considered to be the primary event affecting membrane structure and integrity (Jiang et al., 2007). The composition changes of membrane fatty acids resulting from the decrease in unsaturated fatty acids affected the membrane lipid phase transition (Cao et al., 2011). The Previous reports revealed that the membrane permeability of apple and potato was related to the saturated fatty acids on the membrane (Knowles & Knowles, 1989;Lurie et al., 1987). The increased unsaturated fatty acids in strawberry could help the plant to adapt to low-temperature conditions (Wang & Lin, 2006). 1-MCP combined with heat treatment could inhibit the decrease in linoleic acid and unsaturated-to-saturated fatty acid ratios in loquat fruit (Cao et al., 2009). A high USFA/SFA ratio in the membrane could take advantage of preventing the accumulation of MDA and ion leakage, which could avoid peroxidation and damage (Jin et al., 2014). Previous studies have demonstrated that the biosynthesis of membrane lipids and the biophysical properties of membranes are affected by cellular energy levels (Jiang et al., 2007). It has also been proven that the synthesis of membrane lipids largely depends on ATP synthesis levels in potato cells (Rawyler et al., 1999). In this study, a negative correlation was observed between cellular energy levels and peroxidation of membrane lipids. Furthermore, oxidation of membrane lipids was regarded to occur due to the unstable double bonds of unsaturated fatty acids (Huang et al., 2019). The homeostasis of membrane lipids was largely maintained by the ratio of unsaturated-to-saturated fatty acids. Therefore, the cellular energy status might not be affected by peroxidation of membrane lipids directly. It might be directly affected by the whole status of membrane fatty acids. The ATP level and ATP/ADP were found to be positively correlated with USFA/SFA. Therefore, maintaining or trying to increase the content of unsaturated fatty acids in the membrane could help to protect PA membrane integrity during storage and extend the shelf life of postharvest PA.

| Changes in SDH, CCO, H + -ATPase, and Ca 2+ -ATPase
SDH is a respiration-related enzyme located on the mitochondrial membranes that participated in electron transport chain (ETC) (Kan et al., 2011). As shown in Figure 5a, SDH activity showed downward tendencies in both the 1-MCP treatment and the control. In the first six days, SDH activity in the control was higher than that of the 1-MCP treatment. At the later storage time (9-12 d), SDH activity in the control was significantly lower than that of the 1-MCP treatment (p < .05). This indicated that 1-MCP treatment could effectively inhibit the decline of SDH activity in PA. CCO is regarded as an electron-driven proton pump in respiration process (Zhou et al., 2014). For this reason, electron transport will be affected by the changes in CCO activity and then impact on energy metabolism.
In this study, CCO activity in control declined persistently and higher than that of 1-MCP treatment in the first six days. However, CCO activity in 1-MCP treatment increased slightly and higher than that of the control with significant difference (p < .05) at the later storage time (9-12 d). Activities of H + -ATPase and Ca 2+ -ATPase showed similar decrease trends with those of SDH and CCO. In the first six days, H + -ATPase activity in the control was higher than that of the 1-MCP treatment. At the end of the storage (12 d), H + -ATPase activity in 1-MCP treatment was significantly higher than that of the control (p < .05). Ca 2+ -ATPase activity in 1-MCP treatment was always lower than that of the control during the storage in this study. The difference was significant (p < .05) in the first six days while not significant in 9 d-12 d. Ca 2+ -ATPase activities in both two groups were closed at the end of the storage since Ca 2+ -ATPase activity in 1-MCP treatment increased slightly.
ATP is the main cellular energy resource in eukaryotes, and it is mainly generated from oxidative phosphorylation. Oxidative phosphorylation is an important stage of respiration metabolism that occurring in mitochondria Wu et al., 2016).
As an important part of oxidative phosphorylation, ETC consumes oxygen to generate majority of intracellular energy (Møller, 2001).
The four enzymes (SDH, CCO, H + -ATPase, and Ca 2+ -ATPase) involve in producing ATP by playing important roles in ETC process. SDH involves in ETC process via citric acid cycle and provides electrons to the ubiquinone pool (Luo et al., 2020). CCO is the terminal enzyme of ETC in mitochondria which catalyzes the electrons transport from Ferro cytochrome c to molecule oxygen (Soto et al., 2012). Pumping protons across inner mitochondrial membrane generates proton motive force which is utilized by H + -ATPase to produce ATP (Soole & Menz, 2013). The suppression of H + -ATPase activity may result in the decrease of synthesis rate in ATP (Wei et al., 2018). Ca 2+ -ATPase transports Ca 2+ from cytoplasm to mitochondria to involve in the subsequent oxidation (Luo et al., 2020). Therefore, stable levels of these four enzymes are required to guarantee energy production.
In our study, the activities of SDH, CCO, H + -ATPase, and Ca 2+ -ATPase were maintained even enhanced by 1-MCP treatment. The maintenance of SDH activity might provide a sufficient electron source for ETC. The enhancement of CCO activity might provide a stable electron flow in mitochondria to ensure the generation of proton motive force which was used by H + -ATPase. Although the activity of Ca 2+ -ATPase was lower than that of the control in the later storage time, 1-MCP treatment still showed the maintenance in Ca 2+ -ATPase activity. The results indicated that these enzymes have contributed more production of ATP in 1-MCP treatment. Some similar results were found in previous studies (Jin et al., 2014;Wei et al., 2018).

| NAD + and NADH
The content of NAD + showed significant differences between the 1-MCP treatment and the control except on day 9. The content of NAD + decreased rapidly in the first three days while increased from day 3 to day 12 persistently in the 1-MCP treatment and it was three times than that of the control on day 12. The content of NADH in both two groups showed continuous downward trends. The content of NADH in the control was higher than that of the 1-MCP treatment in the first six days while it was lower than that of the 1-MCP treatment from day 9 to day 12. The results indicated that 1-MCP treatment could maintain the content of NADH and enhance the content of NAD + effectively.
NAD + and NADH are redox pairs in cells. As coenzymes of many redox reactions in organisms, they participate in life activities and transform each other (Birkmayer G D, 2009). NADH plays an important role in maintaining cell growth, differentiation, energy metabolism, and cell protection. Stable level of NADH can provide fuel for the ETC process. The amount of NADH is directly related to ATP production. The more NADH each cell contains, the more energy it produces (Chaban et al., 2014). As shown in Figure 6, the content of NAD + and NADH in the 1-MCP treatment was both significantly higher than those of the control in the later storage time. Since NAD + is oxidized by NADH, the increase of NAD + can prove the energy increase that released by NADH oxidation. In this study, it can be concluded that 1-MCP treatment can inhibit the decrease of NAD + and NADH effectively, and then to maintain the production of ATP and the level of energy metabolism.

| Changes in LOX, LPS, and PLD
As shown in Figure 7a, the activity of LOX in the control increased from day 0 to day 3 and decreased gradually from day 3 to day 12. However, the activity of LOX in the 1-MCP treatment showed obvious fluctuation from day 0 to day 12 and it was higher than that of the control from day 9 to day 12. The activity of LPS in the control decreased persistently during the storage time. The activity of LPS in the 1-MCP treatment decreased from day 0 to day 6 and then increased from day 6 to day 12. It was higher than that of the control in the end. The changes of PLD activity were similar to the pattern of LOX activity. As shown in Figure 7c, the activity of PLD in the control increased to a peak on day 3 and then decreased persistently from day 3 to day 12. The change of PLD activity in the 1-MCP treatment showed a steady trend. At the end of the storage, the activity of PLD in the 1-MCP treatment was higher than that of the control of PA. In this study, the activities of three enzymes were significantly lower than those of the control at the first of the experiment and higher than those of the control at the end of the experiment.
The results indicated that 1-MCP treatment could maintain normal membrane functions via inhibiting the activities of LOX, LPS, and PLD in the early term in PA. It was partially consistent with previous studies (Luo et al., 2020).
In conclusion, 1-MCP was found to possess the ability to delay senescence in PA, which was indicated by the slower decreases in

CO N FLI C T O F I NTE R E S T
No conflict of interest exists in the submission of this manuscript, and manuscript is approved by all authors for publication.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data used to support the findings of this study are included within the article.