Transcriptome Analysis of the Preservation Effect of Three Essential Oil Microcapsules on Okra

: Cinnamon ( Cinnamomum sp.) essential oil microcapsules, oregano ( Origanum sp.) essential oil microcapsules, and oregano–thyme ( Thymus sp.) essential oil microcapsules are rarely used in the postharvest preservation treatment of okra ( Abelmoschus esculentus L.). The mechanism of these three essential oil microcapsules on the postharvest preservation of okra is also not yet well understood. In this study, fresh okra was preserved by three kinds of essential oil microcapsules (cinnamon essential oil microcapsules, oregano essential oil microcapsules, and oregano–thyme essential oil microcapsules). The effect of essential oil microcapsules on the postharvest storage quality of okra was discussed. We also used RNA-Seq to preliminarily explore the mechanism of oregano–thyme essential oil microcapsules on the pre-harvest storage quality of okra. The results showed that the three kinds of essential oil microcapsules could maintain the high sensory evaluation quality and firmness of okra, slow down the increase in respiratory intensity, slow down the total number of colonies on the fruit surface, and slow down weight loss. Through analysis, it was found that the effect of oregano–thyme essential oil microcapsules was remarkably better than that of cinnamon essential oil microcapsules and oregano essential oil microcapsules. The preservation mechanism of oregano–thyme essential oil microcapsules on postharvest okra was preliminarily elucidated by RNA-Seq. This study provides a certain basis for a follow-up study of essential oil microcapsules in the preservation of okra.


Introduction
Okras (Abelmoschus esculentus L.) are grown in areas of southern China such as Fujian [1], although there are also large-scale plantations in India, Iran, and the southern United States [2].We observed in some reports that okra as a crop not only has high economic value, but also possesses some nutrients and active substances with antioxidant and health-promoting effects [3,4].The quality of okras will be reduced due to metabolism and external environmental factors during harvesting and storage.This will affect the edible value of okra [5,6].Preservation measures are usually taken to maintain the storage quality of postharvest okras.Although traditional chemical preservatives have a certain preservation effect on okra, residual preservatives on the surface of the vegetables can cause food safety issues [7].
Many studies have shown that cinnamon (Cinnamomum sp.) essential oil, oregano (Origanum sp.) essential oil, and thyme (Thymus sp.) essential oil have the characteristics of high efficiency, safety, and greenness [8].These three essential oils have antibacterial effects on postharvest fruits and vegetables and can maintain their storage quality [8,9].These three essential oils also have great potential for development as preservatives for fruits and vegetables.For example, applying an amylose coating containing cinnamon essential oil nano lotion to strawberries can alleviate the decline of their storage quality [10].Some researchers found that the use of oregano essential oil on purple skin yams also reached similar conclusions [11].Plum fumigated with thyme essential oil can inhibit browning and related key enzyme activities [12].Similar results were also found in peach [12] and mango [13] studies.A large amount of research is mainly focused on the relationship between the antibacterial effect of essential oil treatment on postharvest fruits and their physiological quality.There is relatively little research on the impact of essential oil treatment on the quality of postharvest okra and the mechanism of its preservation effect.
The low water solubility, high volatility, and strong odor of essential oils limits their application in the preservation of vegetables [14,15].It was found that microencapsulation of essential oils could improve the stability and antioxidant activity of essential oil in vegetable preservation [16,17].Reviewing the literature and previous experiments, we found that β-Cyclodextrin is non-toxic and harmless, and can remarkably improve the essential oil's water solubility and mask its flavor [18,19].The wall material of the essential oil microcapsule used in this experiment is β-Cyclodextrin.
Transcriptome sequencing (RNA-Seq) is currently recognized as a feasible method for analyzing plant gene expression [20,21].In recent years, RNA-Seq has been mostly used to study the mechanism of postharvest preservation of fruits and vegetables [22,23].For example, Wang et al. [24] revealed the influence mechanism of forchlorfenuron (CPPU) treatment on the volatile metabolites and biosynthetic pathways of grapes through RNA-Seq.RNA-Seq analysis also revealed the inhibitory effect of melatonin on ethylene biosynthesis and the factors affecting the quality changes of apples during postharvest storage [25].Jakaria et al. [26] used RNA-Seq to clarify that malate metabolism during postharvest storage of apples is determined by the regulation of NADP malate and phosphoenolpyruvate carboxylase kinase.We found that few studies have applied RNA-Seq to explore the feasibility of essential oil microcapsules to preserve postharvest okra.This study mainly discussed the effects of cinnamon essential oil microcapsules, oregano essential oil microcapsules, and oregano-thyme essential oil microcapsules on the postharvest storage quality of okra.The relatively suitable microencapsulation treatment of essential oil was selected by sensory evaluation and basic indicators, and RNA-Seq revealed the preservation mechanism of essential oil microencapsulation on okra.This experiment provides basic data for further exploring the preservation mechanism of essential oil microcapsules on okra.

Okra
Okras were purchased from Hongqi Agricultural Trade Comprehensive Wholesale Market, Xiqing, Tianjin, China.The okras were transported to the laboratory for selection to remove damaged fruits, and then placed in a fresh-keeping chamber at 5 ± 1 • C for pre-cooling for 16 h.The material used in the experiment was okra fruit.

Treatment of Okra
The pre-cooled okras were selected and graded.The purpose was to remove the okra with mechanical injury and keep the okra maturity consistent.The okras were divided into four treatment groups, and the sample weight of each treatment group was 2 kg.The treatment methods of the four treatment groups are shown in Table 1.Okras were always placed in a foam box in cold storage at 5 ± 1 • C, and samples were taken at 0 days, 3 days, 6 days, 9 days, 12 days, and 15 days, with 0 days as the initial value.

Preparation of Essential Oil Microcapsules
The embedding of the three essential oils was carried out according to the method of Huang et al. [11], and the method was modified.Proportionally, β-Cyclodextrin and sucrose fatty acid ester were added to distilled water at 50 • C, and then the essential oil dissolved in monoglycerides was added in proportion (the ratio of sucrose ester to monoglycerides was 4:1).The solution was emulsified at 40 • C for 30 min and homogenized at 10,000 rpm using a disperser (T25, IKA, Schwabach, Germany) for 3 min.Then, the stable emulsifier was frozen at −55 • C for 24 h.The obtained white powder was an essential oil microcapsule.Monoglyceride, sucrose fatty acid ester, and all three essential oils were purchased from Licang Natural Flavor Oil Co., Ltd.(Ji'an City, China).The microencapsulation process carried out on the three kinds of essential oil microcapsules is shown in Table 2 through preliminary test verification.Emulsifier content, solid content, embedding rate, and emulsion stability tests were repeated 3 times.The embedding of the three essential oils was observed by a Phenom PURE+SED Desktop Scanning Electron Microscope (Phenom-World BV, Eindhoven, The Netherlands).

Sensory Evaluation
According to the method of Falade et al. [27], five professional sensory evaluators were selected to evaluate the degree of color, rot, brittleness, and rusty stains of okra.The sensory evaluation criteria are shown in Table 3. Sensory measurements were performed three times for each treatment group.

Bright green 10
Green 8

Slightly faded 6
Severe fading 4 Yellow 2 The respiratory intensity was measured according to the method of Jia et al. [28].A total of 600 g of okra was placed in a 4500 mL sealed box at 5 ± 1 • C for 1 h.An O 2 /CO 2 gas analyzer (Check Point, Shanghai Jinchuan Mechatronics Technology Co., Ltd., Shanghai, China) was used to measure the respiratory intensity of okra, and the unit of respiratory intensity was mgCO 2 •kg −1 •h −1 .The total plate count on the surface of okras was measured by referring to the methods of Hu et al. [29].A total of 100 g of okras were taken for measurement each time.The unit of total plate count was lgCFU•g −1 .The firmness of okra was measured by a texture analyzer (TA.XT plus, Stable Micro Systems Ltd., Surrey, Godalming, UK).The probe diameter was 2 mm, the puncture depth was 10 mm, the front measurement speed was 2.00 mm s −1 , the measurement speed was 0.50 mm s −1 , and the post-measurement speed was 2.00 mm s −1 .The unit of firmness was kg cm −2 .The weight loss rate of okra was measured according to the method of Jia et al. [28].During each sampling, the fixed fruits of the treatment group were weighed.The result of the weight loss rate is the percentage.Three parallel determinations were carried out in the above experiments, and the results were averaged.

RNA Extraction, RNA-Seq Library Construction, and Data Analysis
Okra samples were sent to a third-party testing company for transcriptome analysis.According to the detection method provided by a third party, total RNA was extracted by a Tiangen polysaccharide polyphenol Kit (QIAGEN, Hilden, Germany), RNA integrity was tested with an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA), and subsequent tests were continued for samples with RNA integrity value (RIN) ≥7.According to the method provided by a third party, the trustee stranded mRNA sample prep Kit (Illumina, San Diego, CA, USA) was used for library construction.The library was sequenced on the Illumina novaseq 6000 (Illumina, San Diego, CA, USA) sequencing platform and 150 bp paired-end reads were generated.Trimmomatic software (version 0.36) was used to control the quality of sequencing data.Trinity (v2.6.6)software was used to splice clean reads, and Unigenes were selected according to sequence similarity and bp number.Blast2GO [30] was used to annotate the gene functions of single genes in databases (NR, NT, PFAM, KOG, and Swiss prot).To obtain the GO annotations of Unigenes according to the method of Sun et al. [31], the Unigenes were compared with the KEGG database to obtain channel information.Bowtie2 and eXpress software (version number: 1.5.1)were used for FPKM and count analysis.Three groups of parallel measurements were set in the test.

Statistical Analysis of the Results
The indicators of each treatment group were measured three times.The data obtained in the test are expressed in the form of mean ± standard deviation (SD).Origin2022 (origin lab, Northampton, MA, USA) was used to draw the statistical map; IBM SPSS statistics (Version 22.0, Chicago, IL, USA) was used for statistical analysis; and Tukey's test was used to analyze the significant differences between the data, with a confidence level of p < 0.05.The heat map was drawn by TBtools.The volcano map, Venn diagram, enrichment analysis image, and classification statistical map were drawn by R (Version 3.5.0).

Scanning Electron Microscope Observation of Microencapsulation of Three Essential Oils
The embedding of microcapsules of three essential oils is shown in Figure 1.Irregular crystals of different sizes can be observed through the pictures.The presence of large crystals proved the formation of a complex.Similar results were obtained by Shah et al. [32].Prabu et al. and Wang et al. also obtained similar results [33,34].The morphological characteristics shown in the picture prove the formation of microcapsules [35].This indicates that the three microcapsules can be used in subsequent experiments.

Statistical Analysis of the Results
The indicators of each treatment group were measured three times.The data obtained in the test are expressed in the form of mean ± standard deviation (SD).Origin2022 (origin lab, Northampton, MA, USA) was used to draw the statistical map; IBM SPSS statistics (Version 22.0, Chicago, IL, USA) was used for statistical analysis; and Tukey's test was used to analyze the significant differences between the data, with a confidence level of p < 0.05.The heat map was drawn by TBtools.The volcano map, Venn diagram, enrichment analysis image, and classification statistical map were drawn by R (Version 3.5.0).

Scanning Electron Microscope Observation of Microencapsulation of Three Essential Oils
The embedding of microcapsules of three essential oils is shown in Figure 1.Irregular crystals of different sizes can be observed through the pictures.The presence of large crystals proved the formation of a complex.Similar results were obtained by Shah et al. [32].Prabu et al. and Wang et al. also obtained similar results [33,34].The morphological characteristics shown in the picture prove the formation of microcapsules [35].This indicates that the three microcapsules can be used in subsequent experiments.
Figure 1.Scanning electron microscopy images of essential oil microcapsules at 10,000x magnification, CEO is cinnamon essential oil microcapsules, OEO is oregano essential oil microcapsules, and OTEO is oregano-thyme essential oil microcapsules.

Effects of Microencapsulation of Different Essential Oils on Sensory Evaluation Quality of Okra
The sensory characteristics of fruits and vegetables will directly affect consumers' purchase desire [36,37].During storage, the sensory qualities of okra will change due to its respiratory metabolism and other factors.These changes include the gradual yellowing of the fruit color, the gradual appearance of rust spots on the fruit surface, a decline in firmness, and decay [38].The changes in the sensory quality of okras during storage are shown in Figure 2.During storage, the sensory evaluation scores of color, rot, brittleness, and rusty stains of okra showed a downward trend as a whole.The scores of four sensory evaluation indexes in the OTEO (oregano-thyme essential oil microcapsules) treatment group were higher than those in the control treatment group, CEO (cinnamon essential oil microcapsules) treatment group, and OEO (oregano essential oil microcapsules) treatment group.This indicated that OTEO treatment had a positive effect on delaying the reduction in okra quality during storage.During storage, the color of the okras in the OTEO treatment group remained green, while the okras in the control treatment group began to show a slight fading of color by 9 days of storage, fading seriously by 15 days.The rotted area of the okras in each treatment group gradually increased during storage.At the end of storage, the rotted area of okras in the OTEO treatment group was less than

Effects of Microencapsulation of Different Essential Oils on Sensory Evaluation Quality of Okra
The sensory characteristics of fruits and vegetables will directly affect consumers' purchase desire [36,37].During storage, the sensory qualities of okra will change due to its respiratory metabolism and other factors.These changes include the gradual yellowing of the fruit color, the gradual appearance of rust spots on the fruit surface, a decline in firmness, and decay [38].The changes in the sensory quality of okras during storage are shown in Figure 2.During storage, the sensory evaluation scores of color, rot, brittleness, and rusty stains of okra showed a downward trend as a whole.The scores of four sensory evaluation indexes in the OTEO (oregano-thyme essential oil microcapsules) treatment group were higher than those in the control treatment group, CEO (cinnamon essential oil microcapsules) treatment group, and OEO (oregano essential oil microcapsules) treatment group.This indicated that OTEO treatment had a positive effect on delaying the reduction in okra quality during storage.During storage, the color of the okras in the OTEO treatment group remained green, while the okras in the control treatment group began to show a slight fading of color by 9 days of storage, fading seriously by 15 days.The rotted area of the okras in each treatment group gradually increased during storage.At the end of storage, the rotted area of okras in the OTEO treatment group was less than 1/5.The rotted area of okras in the control treatment group was more than 1/5 at the end of storage.The brittleness of okras decreased gradually during storage.The brittleness scores of each treatment group were the same before 3 days of storage, and then with the extension of storage time, the brittleness of okra in each treatment group gradually began to differ.At the end of storage, okras in the control treatment group showed a very soft state.Okras in the OTEO treatment group were broken in half at the end of storage.The rusty stains on the surface of the okras gradually increased during storage.At the end of storage, the surface of okras in the control treatment group showed large rusty stains and depression.There were only a few brown spots on the surface of okras in the OTEO treatment group.Through sensory evaluation, we found that CEO, OEO, and OTEO treatments had preservation effects on okra.The preservation effect of OTEO treatment on okra storage was better than CEO treatment, OEO treatment, and control treatment.This was because OTEO treatment remarkably slowed down the respiratory metabolism of okra during storage, and its bactericidal effect on the surface of okra was also more obvious.Zhang et al. [39] also found that soaking citrus in a compound solution of lemon essential oil, chitosan, and calcium chloride can maintain the sensory quality of citrus.It has also been observed that the use of chitosan coatings containing tarragon essential oil on kumquat can maintain the sensory qualities of kumquat [40].Similar test conclusions were also confirmed on fresh-cut loquat [41], fresh-cut apricot [42], and fresh-cut orange fruit [43].Through comprehensive analysis, OTEO treatment has a positive effect on delaying the reduction in sensory qualities of postharvest okra during storage, and the preservation effect is better than CEO treatment and OEO treatment.
Horticulturae 2024, 10, x FOR PEER REVIEW 6 of 15 1/5.The rotted area of okras in the control treatment group was more than 1/5 at the end of storage.The brittleness of okras decreased gradually during storage.The brittleness scores of each treatment group were the same before 3 days of storage, and then with the extension of storage time, the brittleness of okra in each treatment group gradually began to differ.At the end of storage, okras in the control treatment group showed a very soft state.Okras in the OTEO treatment group were broken in half at the end of storage.The rusty stains on the surface of the okras gradually increased during storage.At the end of storage, the surface of okras in the control treatment group showed large rusty stains and depression.There were only a few brown spots on the surface of okras in the OTEO treatment group.Through sensory evaluation, we found that CEO, OEO, and OTEO treatments had preservation effects on okra.The preservation effect of OTEO treatment on okra storage was better than CEO treatment, OEO treatment, and control treatment.This was because OTEO treatment remarkably slowed down the respiratory metabolism of okra during storage, and its bactericidal effect on the surface of okra was also more obvious.Zhang et al. [39] also found that soaking citrus in a compound solution of lemon essential oil, chitosan, and calcium chloride can maintain the sensory quality of citrus.It has also been observed that the use of chitosan coatings containing tarragon essential oil on kumquat can maintain the sensory qualities of kumquat [40].Similar test conclusions were also confirmed on fresh-cut loquat [41], fresh-cut apricot [42], and fresh-cut orange fruit [43].Through comprehensive analysis, OTEO treatment has a positive effect on delaying the reduction in sensory qualities of postharvest okra during storage, and the preservation effect is better than CEO treatment and OEO treatment.The respiration intensity of okras during postharvest storage is shown in Figure 3A.The respiratory intensity of okras first increased and then decreased during storage.The respiratory peak of okras occurred on the 9th day.During the storage of okras, the respiratory intensity of each treatment group was remarkably lower than that of the control treatment group (p < 0.05).This was due to the inhibitory effect of essential oil treatment on the respiration of okras.In each treatment group, the respiratory intensity of the OTEO treatment group was remarkably lower than that of the OEO treatment group and CEO treatment group (p < 0.05).This was because OETO treatment was more remarkable in inhibiting the respiratory intensity of okras during postharvest storage.Shehata et al. [44] pointed out that citrus essential oil can remarkably reduce the respiratory intensity of strawberries during storage.Similar studies have been conducted by soaking figs in chitosan-containing thyme essential oil [45].During the postharvest storage of vegetables, they will decay due to microbial action on the fruit surface [46].The total plate count on the surface of okra fruits during postharvest storage is shown in Figure 3B.The total plate count on the surface of okra shows a gradually increasing trend during storage.The total plate count in the control treatment group was remarkably higher than in the treatment group (p < 0.05).This was because essential oil treatment has a certain bactericidal effect on the surface of okras, resulting in a lower total plate count.The total plate count in the OTEO treatment group was remarkably lower than in the OEO and CEO treatment groups (p < 0.05).This was due to the stronger bactericidal ability of OTEO treatment.This also conforms with the results obtained from the decay index in sensory evaluation.Martínez et al. [47] pointed out that using chitosan coatings containing thyme essential oil on the surface of strawberries can significantly reduce the total plate count.Lettuce was treated with thyme essential oil embedded with β-Cyclodextrin microcapsules, which also remarkably reduced the total plate count on the surface of lettuce after treatment [48].Okra softens during storage due to its ripening and respiratory metabolism, leading to changes in its properties [49].As shown in Figure 3C, the firmness of okras showed a gradually decreasing trend during postharvest storage, which was due to the gradual decrease in okra firmness caused by respiratory metabolism during storage.The firmness of the control treatment group was remarkably lower than that of the other three treatment groups (p < 0.05).This was because the treatment of essential oil microcapsules has a slowing effect on the decrease in firmness of okras.The firmness of okras in the OTEO treatment group was remarkably higher than that in the CEO and OEO treatment groups (p < 0.05).This indicated that OTEO treatment could delay the decline of okra firmness.At the same time, this is also in line with the results of the brittleness index in sensory evaluation.Adriana C et al. [50] also found that strawberries soaked with polysaccharide coating containing eugenol essential oil can remarkably alleviate the decline of its firmness.Other studies have also observed that oregano essential oil β-Cyclodextrin can also delay the decline in firmness of purple skin yams [11].
Factors such as respiratory metabolism and microbial reproduction during postharvest storage can lead to an increase in vegetable weight loss rate [51,52].As shown in Figure 3D, the weight loss rate of okras showed a gradually increasing trend during postharvest storage, and there was no remarkable difference between the control treatment group and the essential oil treatment group in the first 3 days (p > 0.05).With the extension of storage time, there were remarkable differences between the treatment groups.From the 3rd day onwards, the weight loss rate of the control treatment group was remarkably higher than that of the OTEO treatment group, OEO treatment group, and CEO treatment group (p < 0.05).The weight loss rate of the OTEO treatment group was remarkably lower than that of the CEO treatment group and OEO treatment group (p < 0.05).This was because the essential oil treatment has a delaying effect on the increase in the weight loss rate of okras during storage, and the delaying effect of the OTEO treatment is more obvious than the CEO treatment and OEO treatment.Other studies have also observed that the use of an edible basil gum coating containing oregano essential oil to treat fresh-cut apricots can delay the increase in weight loss rate [42].A similar conclusion was reached using an edible pectin coating containing orange peel essential oil to treat fresh-cut oranges [43].

RNA Sequencing Results
According to the above test results, it was determined that the OTEO microencapsulation treatment has a delaying effect on the deterioration of the storage quality of postharvest okra.To further clarify the mechanism of OTEO microcapsule treatment on postharvest okra, we selected the control-treated group and the OTEO-treated group at 9 days for RNA-Seq sequencing analysis.Three biological replicates were performed for each treatment group.A total of 37.8 G data was obtained by sequencing.GC was 43.82-45%, Q20 was 97.47-98.27%,and Q30 was 92.83-94.62%.The summary of the sequencing quality is shown in Table S1.

Annotation of Gene Function
We annotated Unigene concerning five databases: NR, NT, Pfam, GO, and KOG.The annotation results are shown in Table S2.In total, 67,765, 62,250, 42,365, 42,362, and 15,977 transcripts were detected in the NR, NT, Pfam, GO, and KOG databases, respectively.According to the Venn diagram (Figure S1), there were 10,923 annotated Unigenes; 5185 and 5843 Unigenes were annotated separately in NT and NR, respectively, while only one Unigene in Pfam was annotated separately.Among the 67,765 Unigenes found and annotated in the NR database, Gossypium barbadense (19.8%),Gossypium raimondii (14.8%),

RNA Sequencing Results
According to the above test results, it was determined that the OTEO microencapsulation treatment has a delaying effect on the deterioration of the storage quality of postharvest okra.To further clarify the mechanism of OTEO microcapsule treatment on postharvest okra, we selected the control-treated group and the OTEO-treated group at 9 days for RNA-Seq sequencing analysis.Three biological replicates were performed for each treatment group.A total of 37.8 G data was obtained by sequencing.GC was 43.82-45%, Q20 was 97.47-98.27%,and Q30 was 92.83-94.62%.The summary of the sequencing quality is shown in Table S1.

Gene Expression
We used the FPKM method to calculate the Unigene expression levels of six samples.To ensure the reliability of the test data, we performed three biological replicates for each treatment group.The Encode plan recommends that the square of the Pearson correlation coefficient (R 2 ) be greater than 0.92 (under ideal sampling and experimental conditions) [53].According to the results of the analysis (Figure 4), the R 2 between biological replicates was greater than 0.8.This indicates that the sequencing data obtained by RNA-Seq are reliable and can be used for subsequent analysis.

Gene Expression
We used the FPKM method to calculate the Unigene expression levels of six samples.To ensure the reliability of the test data, we performed three biological replicates for each treatment group.The Encode plan recommends that the square of the Pearson correlation coefficient (R 2 ) be greater than 0.92 (under ideal sampling and experimental conditions) [53].According to the results of the analysis (Figure 4), the R 2 between biological replicates was greater than 0.8.This indicates that the sequencing data obtained by RNA-Seq are reliable and can be used for subsequent analysis.

Differential Gene Expression Analysis
We compared the control group and the OTEO treatment group on the 9th day and identified a total of 4813 DEGs, of which 2293 DEGs were up-regulated and 1820 DEGs were down-regulated.According to the volcano diagram (Figure 5), it can be intuitively seen that the number of up-regulated DEGs is greater than that of down-regulated DEGs, indicating that OTEO treatment has an up-regulation effect on okra during storage.

Differential Gene Expression Analysis
We compared the control group and the OTEO treatment group on the 9th day and identified a total of 4813 DEGs, of which 2293 DEGs were up-regulated and 1820 DEGs were down-regulated.According to the volcano diagram (Figure 5), it can be intuitively seen that the number of up-regulated DEGs is greater than that of down-regulated DEGs, indicating that OTEO treatment has an up-regulation effect on okra during storage.We performed a GO enrichment analysis on the top 20 DEGs of significance.The enrichment results are shown in Figure 6.Among the 2621 DEGs in the enrichment analysis, 1577 DEGs were up-regulated and 1044 DEGs were down-regulated, which mapped to a total of 133 terms, 64 biological processes, 37 molecular functions, and 31 cellular components.As shown in Figure 6, DEGs in biological processes are mainly enriched in biosynthetic processes, carbohydrate metabolic processes, ribosome biogenesis, and the generation of precursor metabolites and energy.DNA binding transcription factor activity, oxidoreductase activity and structural constitution of ribosome were remarkably enriched in molecular functions, while protein-containing complexes and ribosomes were remarkably enriched in the functions of cellular components.After the KEGG metabolic pathways of DEGs were enriched (Figure 7), the enrichment was mainly concentrated in the ribosome, biosynthesis of secondary metabolites, metabolic pathways, plant hormone signal transduction, glycolysis/gluconeogenesis, MAPK signaling pathway-plant, alphalinolenic acid metabolism, phenylpropanoid biosynthesis, and other aspects.The results of the GO enrichment analysis showed that the effects of OTEO essential oil on the storage quality of okras mainly involved biosynthetic processes, molecular functions, and cellular components.This also corresponds to the results of respiratory intensity and weight loss rate.This is because plants need energy for respiration and metabolism, and most of the time they need carbohydrates as the substrate for their respiration.The synthesis and metabolism of organisms will also be manifested through mass reduction [54][55][56].Plant hormone signal transduction, phenylpropanoid biosynthesis, and MAPK signaling We performed a GO enrichment analysis on the top 20 DEGs of significance.The enrichment results are shown in Figure 6.Among the 2621 DEGs in the enrichment analysis, 1577 DEGs were up-regulated and 1044 DEGs were down-regulated, which mapped to a total of 133 terms, 64 biological processes, 37 molecular functions, and 31 cellular components.As shown in Figure 6, DEGs in biological processes are mainly enriched in biosynthetic processes, carbohydrate metabolic processes, ribosome biogenesis, and the generation of precursor metabolites and energy.DNA binding transcription factor activity, oxidoreductase activity and structural constitution of ribosome were remarkably enriched in molecular functions, while protein-containing complexes and ribosomes were remarkably enriched in the functions of cellular components.After the KEGG metabolic pathways of DEGs were enriched (Figure 7), the enrichment was mainly concentrated in the ribosome, biosynthesis of secondary metabolites, metabolic pathways, plant hormone signal transduction, glycolysis/gluconeogenesis, MAPK signaling pathway-plant, alpha-linolenic acid metabolism, phenylpropanoid biosynthesis, and other aspects.The results of the GO enrichment analysis showed that the effects of OTEO essential oil on the storage quality of okras mainly involved biosynthetic processes, molecular functions, and cellular components.This also corresponds to the results of respiratory intensity and weight loss rate.This is because plants need energy for respiration and metabolism, and most of the time they need carbohydrates as the substrate for their respiration.The synthesis and metabolism of organisms will also be manifested through mass reduction [54][55][56].Plant hormone signal transduction, phenylpropanoid biosynthesis, and MAPK signaling pathway-plant in KEGG enrichment analysis of plants are the most significant enrichment pathways of plant disease resistance [57,58].This also confirms the results that OTEO treatment has an inhibitory effect on okra decay and the total number of colonies.Through GO estimation analysis and KEGG enrichment analysis, we found that OTEO treatment could maintain a higher storage quality of postharvest okras.This result also aligned with the conclusions drawn from sensory evaluation, respiratory intensity, total plate count, and firmness.Jiang et al. [57] used eugenol essential oil to treat grapes and performed transcriptome GO and KEGG enrichment analysis and the results were similar to the results of this experiment.Wei et al. [59] used tea tree oil essential oil to treat pre-harvest strawberries.After KEGG enrichment analysis of the samples, they also found significant enrichment in plant hormone signal transduction and linolenic acid metabolism pathways.
pathway-plant in KEGG enrichment analysis of plants are the most significant enrichment pathways of plant disease resistance [57,58].This also confirms the results that OTEO treatment has an inhibitory effect on okra decay and the total number of colonies.Through GO estimation analysis and KEGG enrichment analysis, we found that OTEO treatment could maintain a higher storage quality of postharvest okras.This result also aligned with the conclusions drawn from sensory evaluation, respiratory intensity, total plate count, and firmness.Jiang et al. [57] used eugenol essential oil to treat grapes and performed transcriptome GO and KEGG enrichment analysis and the results were similar to the results of this experiment.Wei et al. [59] used tea tree oil essential oil to treat preharvest strawberries.After KEGG enrichment analysis of the samples, they also found significant enrichment in plant hormone signal transduction and linolenic acid metabolism pathways.

Conclusions
The experimental results showed that three kinds of essential oil microcapsules could inhibit the deterioration of the sensory evaluation qualities of okra after harvest.Compared with the control, the oil microcapsule treatment inhibited the increase in okra respiration intensity and the total number of colonies on the fruit surface during storage and

Conclusions
The experimental results showed that three kinds of essential oil microcapsules could inhibit the deterioration of the sensory evaluation qualities of okra after harvest.Compared with the control, the oil microcapsule treatment inhibited the increase in okra respiration intensity and the total number of colonies on the fruit surface during storage and maintained the higher hardness of okra.The microcapsule treatment of essential oils delayed the rise in the weight loss rate of okra during storage.The effect of OTEO treatment was better than CEO treatment and OEO treatment.Through transcriptomic sequencing of okra samples in the OTEO treatment group on the 9th day, we preliminarily clarified the preservation mechanism of OETO treatment on postharvest okra.The comprehensive test results showed that the three kinds of essential oil microcapsules had a preservation effect on postharvest okra.The fresh-keeping effect of OTEO was better than CEO and OEO.The experimental results can provide help for further exploring the effect of microencapsulation treatment of essential oil on the postharvest preservation of okra.In the future, we will continue to explore the feasibility of essential oil microcapsules on the preservation of postharvest okra by combining proteomics and other technologies.

Figure 1 .
Figure 1.Scanning electron microscopy images of essential oil microcapsules at 10,000× magnification, CEO is cinnamon essential oil microcapsules, OEO is oregano essential oil microcapsules, and OTEO is oregano-thyme essential oil microcapsules.

Figure 2 .
Figure 2. Effect of different essential oil microencapsulation treatments on okra color, rot, brittleness, and rusty stains.3.3.Effects of Different Essential Oil Microcapsules on the Respiration Intensity, Total Plate Count, Firmness, and Weight Loss Rate of OkraThe respiration intensity of okras during postharvest storage is shown in Figure3A.The respiratory intensity of okras first increased and then decreased during storage.The respiratory peak of okras occurred on the 9th day.During the storage of okras, the respiratory intensity of each treatment group was remarkably lower than that of the control

Figure 2 .
Figure 2. Effect of different essential oil microencapsulation treatments on okra color, rot, brittleness, and rusty stains.

3. 3 .
Effects of Different Essential Oil Microcapsules on the Respiration Intensity, Total Plate Count, Firmness, and Weight Loss Rate of Okra

Figure 3 .
Figure 3. Effects of different essential oil microencapsulation treatments on the respiratory intensity (A), total number of colonies (B), firmness (C), and weight loss rate (D) of okra.The lowercase letters (a-d) in the figure indicate the significant differences between different essential oil microencapsulation treatments for the same storage days (p < 0.05).

Figure 3 .
Figure 3. Effects of different essential oil microencapsulation treatments on the respiratory intensity (A), total number of colonies (B), firmness (C), and weight loss rate (D) of okra.The lowercase letters (a-d) in the figure indicate the significant differences between different essential oil microencapsulation treatments for the same storage days (p < 0.05).

Figure 4 .
Figure 4. Heat map of correlation coefficient between samples.The abscissa is lg (FPKM+1) of sample 1, and the ordinate is lg (FPKM+1) of sample 2. R 2 : The square of Pearson correlation coefficient.

Figure 4 .
Figure 4. Heat map of correlation coefficient between samples.The abscissa is lg (FPKM+1) of sample 1, and the ordinate is lg (FPKM+1) of sample 2. R 2 : The square of Pearson correlation coefficient.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/horticulturae10020193/s1,FigureS1: Venn diagram; FigureS2: Species distribution map of NR library ratio pairs of okra Unigene sequence, FigureS3: GO annotation classification statistics of okra Unigene sequence; FigureS4: KOG annotation classification statistical map of okra Unigene sequence; FigureS5: KEGG metabolic pathway classification statistics of okra Unigene sequence; TableS1: Summary of sequencing quality; TableS2: gene annotation results.Author Contributions: S.J.: conceptualization, writing-original draft, software.H.Z. revised and adjusted the content of the paper.Q.Q.: resources, data curation, validation.S.Y.: investigation, formal analysis, supervision.C.C.: supervision, writing-review.L.L.: funding acquisition, project administration.This paper was written with full disclosure of all the potential conflicts of interest by all the authors, who reviewed and approved the final text.All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by the Key Project of Tianjin Natural Science Foundation (20JCZDJC00420); National Natural Science Foundation of China: Study on the regulatory mechanism of AP2/EREBP transcription factor in the formation of heart browning after Yali pear harvest (32072278); Tianjin Science and Technology Program Project (22ZYCGSN00170, 22ZYCGSN00470); Yingkou Enterprise Doctoral Shuangchuang Program Project (2022NYNS013); Jining Key Research and Development Program Project (2022NYNS013); Tianjin Forestry and Fruit Modern Agricultural Industrial Technology System Innovation Team Project (ITTHRS2021000); and Gansu Provincial Science and Technology Program Project East-West Collaboration Special "Winter fruit pear green storage and preservation and quality control technology demonstration and promotion" (23CXNA0026).Data Availability Statement: Data are contained within the article.

Table 2 .
Microencapsulation process of three essential oils.

Table 3 .
Sensory evaluation criteria of okra.