Transcriptome Analysis of ‘Haegeum’ Gold Kiwifruit Following Ethylene Treatment to Improve Postharvest Ripening Quality

Fruit ripening involves changes in physical, physiological and metabolic activities through the actions of enzymes and regulatory genes. This study was initiated to identify the genes related to the ripening of kiwifruit. Gold ‘Haegeum’ kiwifruit is a yellow-fleshed kiwifruit cultivar usually used for fresh marketing. The fruit is harvested at a physiologically mature but unripe stage for proper storage, marketing distribution and longer shelf life. To identify the differentially expressed genes (DEGs) during ripening, fruit treated with ethylene were compared with control fruit that ripened naturally without ethylene treatment. Firmness, respiration rate, ethylene production rate, total soluble solids (TSS), titratable acidity (TA), brix acid ratio (BAR) and overall acceptability were taken during the study as fruit ripening indicators. Total mRNAs were sequenced by Illumina high-throughput sequencing platform and the transcriptome gene set was constructed by de novo assembly. We identified 99,601 unigenes with an average length of 511.77 bp in transcriptome contigs. A total of 28,582 differentially expressed unigenes were identified in the ethylene treatment vs. control. Of these 28,582 unigenes, 13,361 and 15,221 genes were up- and downregulated, respectively, in the treated fruit. The results also showed that 1682 and 855 genes were up- and downregulated, respectively, more than 2-fold at p < 0.05 in fruit treated with ethylene as compared with the control fruit. Moreover, we identified 75 genes showing significantly different expression; 42 were upregulated, and 33 were downregulated. A possible category of the identified ripening-related genes was also made. The findings of this study will add to the available information on the effect of ethylene treatment on ripening and the related changes of kiwifruit at the genomic level, and it could assist the further study of genes related to ripening for kiwifruit breeding and improvement.


Introduction
Kiwifruit (Actinidia spp.) is a perennial deciduous warm-temperate fruit that belongs to the family Actinidiaceae, and it is native to Northern China, Korea, Siberia, and Japan [1]. According to FAOSTAT [2], worldwide annual production of kiwifruit was 4.32 million Mt from the total planted We identified 99,274 unigenes with an average length of 513.5 bp in transcriptome contigs. The average unigene length for kiwifruit in this study was comparable to those observed in other species such as persimmon (643.2 bp) [18], Oncidium (493 bp) [28], and Pinus contorta (500 bp) [29]. The identified unigenes were classified into three functional categories; 45,275 genes were assigned to biological processes, 8223 to molecular function and 13,224 to the cellular component. Further subcategories were also observed within each main category of the GO classification scheme and the dominant 10 subcategories from each main category are presented in Figure 1. Kiwifruit annotation in the present study gained more descriptive information (67.2%) than those observed in other species such as Diospyros kaki (61.1%) [18], Bupleurum chinense (52.6%) [30] and Dendrocalamus latiflorus (54.9%) [31]. 4 Results of the considered quality parameters were expressed as means ± standard errors. SAS statistical software (SAS/STAT 9.1; SAS Institute Inc., USA) was implemented for statistical analyses, and tests for significance were done using t-test.

Assembly and Annotation
The summaries of the mapping and sequencing results are presented in Tables 1 and 2. The RNA  sequence analysis was made by using Illumina Truseq Stranded mRNA library prep kit and Novaseq 6000  for sequencing. A total of 51,331,356 and 50,615,434 reads were generated from the control and ethylene  libraries, with mapped read numbers of 41,747,134 and 41,801,951, respectively (Table 1). We identified 99,274 unigenes with an average length of 513.5 bp in transcriptome contigs. The average unigene length for kiwifruit in this study was comparable to those observed in other species such as persimmon (643.2 bp) [18], Oncidium (493 bp) [28], and Pinus contorta (500 bp) [29]. The identified unigenes were classified into three functional categories; 45,275 genes were assigned to biological processes, 8223 to molecular function and 13,224 to the cellular component. Further subcategories were also observed within each main category of the GO classification scheme and the dominant 10 subcategories from each main category are presented in Figure 1. Kiwifruit annotation in the present study gained more descriptive information (67.2%) than those observed in other species such as Diospyros kaki (61.1%) [18], Bupleurum chinense (52.6%) [30] and Dendrocalamus latiflorus (54.9%) [31].

DEGs in the Comparison of Ethylene-Treated vs. Control Kiwifruit
A total of 28,582 unigenes were differentially expressed during the comparison of ethylene-treated vs. control 'Haegeum' gold kiwifruit. From the differentially expressed unigenes, 13,361 were upregulated and 15,221 were downregulated in the ethylene-treated fruit. The number of genes that induced expression of more than 2-fold change at p < 0.05 was 1682, and the number of genes that revealed inhibited expression of more than 2-fold change at p < 0.05 was 855 ( Table 3). The mainly expressed genes during ethylene treatment of 'Haegeum' gold kiwifruit were the genes involved in cellular and metabolic process, the genes involved in response to stimulus in the biological process, the genes involved in binding and catalytic activities in the molecular function and the genes involved in cell and cell parts in the cellular component (Figures 2-4).

DEGs in the Comparison of Ethylene-Treated vs. Control Kiwifruit
A total of 28,582 unigenes were differentially expressed during the comparison of ethylene-treated vs. control 'Haegeum' gold kiwifruit. From the differentially expressed unigenes, 13,361 were upregulated and 15,221 were downregulated in the ethylene-treated fruit. The number of genes that induced expression of more than 2-fold change at p < 0.05 was 1682, and the number of genes that revealed inhibited expression of more than 2-fold change at p < 0.05 was 855 ( Table 3). The mainly expressed genes during ethylene treatment of 'Haegeum' gold kiwifruit were the genes involved in cellular and metabolic process, the genes involved in response to stimulus in the biological process, the genes involved in binding and catalytic activities in the molecular function and the genes involved in cell and cell parts in the cellular component (Figures 2-4).

Firmness and Genes related to Softening
Ethylene treatment efficiently hastens the ripening process as observed on the firmness ( Figure 5). The firmness in the treated 'Haegeum' gold kiwifruit decreased sharply from 26.99 N at harvest to 4.82 N on day 3, in contrast to 23.01 N in control fruit on the same day ( Figure 5). Table 4 shows the expression levels of the significantly upregulated unigenes involved in the ripening of kiwifruit. Physiological processes are associated with the complex fruit softening processes and result in the remodeling of cell wall structure

Firmness and Genes related to Softening
Ethylene treatment efficiently hastens the ripening process as observed on the firmness ( Figure 5). The firmness in the treated 'Haegeum' gold kiwifruit decreased sharply from 26.99 N at harvest to 4.82 N on day 3, in contrast to 23.01 N in control fruit on the same day ( Figure 5). Table 4 shows the expression levels of the significantly upregulated unigenes involved in the ripening of kiwifruit. Physiological processes are associated with the complex fruit softening processes and result in the remodeling of cell wall structure [32]. Cell wall hydrolytic enzymes such as polygalacturonase (PG), pectate lyase (PL), pectin acetylesterase and β-galactosidase (β-gal) are involved in the ripening process by solubilizing pectin polysaccharides of fruits [33][34][35][36]. Interestingly, in this study, all PG, PL, pectin acetylesterase and β-gal genes were upregulated due to ethylene treatment, in turn hastening the action of pectin-digesting enzymes that lead to softening of kiwifruit. Besides, xyloglucan endotransglucosylase/hydrolase and oligopeptide transporter-like were upregulated in our study. According to Schroder and Atkinson [37], xyloglucans are thought to be part of a cellulose-hemicellulose framework that stabilizes the cell wall. So, the upregulation of the xyloglucan endotransglucosylase/hydrolase gene could lead to the ripening and softening of kiwifruit. The upregulation of an oligopeptide transporter-like which encodes integral membrane proteins to translocate substrates into the cytosol [38] could be related to the solubilization of pectin polysaccharides during ripening. Furthermore, β-1, 4-mannosyl-glycoprotein-like which is involved in cell wall modification and degradation [39] and cinnamoyl CoA-reductase (CCR), an enzyme that catalyzes key steps in the biosynthesis of lignin, were also upregulated due to ethylene treatment. Consistent with this study, Li et al. [40] reported the increase in lignin content during cold storage implying the fact that stimulation of lignin biosynthesis could be due to stresses like low temperature and exogenous application of ethylene.
An increased amount of trehalose, nonreducing disaccharide, in fruits has been reported to improve shelf life [41]. In this study, the gene that encodes trehalase (α-glucoside-1-glucohydrolase), a hydrolytic cell wall-bound enzyme that cleaves trehalose into two glucose moieties [41] was upregulated due to ethylene treatment. This phenomenon degrades apoplastic trehalose which in turn causes softening of the fruit ( Figure 5).
Contrarily, the genes that encode cellulose synthase A catalytic subunit 8 (UDP-forming)-like, which takes part in synthesizing cellulose [42], expansin-A1-like, cell wall proteins that affect cell wall composition [43] were downregulated implying that catabolic process could dominate the anabolic process during ripening of fruits. Mannan endo-1,4-beta-mannosidase, which is involved in the cell wall hydrolysis [44], and caffeoylshikimate esterase (CSE), which plays an essential role in lignin biosynthesis [45], were also downregulated due to ethylene treatment probably due to full ripening of kiwifruit before 3 days after treatment with exogenous ethylene. In addition, the genes that encode aquaporin PIP1-4-like and aquaporin PIP2-7-like were downregulated probably due to the reduction of the transport of water and solutes across the cell membrane [46] as the fruit ripened. 7 wall-bound enzyme that cleaves trehalose into two glucose moieties [41] was upregulated due to ethylene treatment. This phenomenon degrades apoplastic trehalose which in turn causes softening of the fruit ( Figure 5).
Contrarily, the genes that encode cellulose synthase A catalytic subunit 8 (UDP-forming)-like, which takes part in synthesizing cellulose [42], expansin-A1-like, cell wall proteins that affect cell wall composition [43] were downregulated implying that catabolic process could dominate the anabolic process during ripening of fruits. Mannan endo-1,4-beta-mannosidase, which is involved in the cell wall hydrolysis [44], and caffeoylshikimate esterase (CSE), which plays an essential role in lignin biosynthesis [45], were also downregulated due to ethylene treatment probably due to full ripening of kiwifruit before 3 days after treatment with exogenous ethylene. In addition, the genes that encode aquaporin PIP1-4-like and aquaporin PIP2-7-like were downregulated probably due to the reduction of the transport of water and solutes across the cell membrane [46] as the fruit ripened.

Ethylene Production and Respiration Rates and Related Genes
As observed in Figure 5, respiration and ethylene production rates were also higher on the third day in ethylene-treated fruit as compared with the control. The final step in ethylene biosynthesis is catalyzed by 1-aminocyclopropane-1-carboxylate oxidase (ACCO) [18,47]. We observed significant upregulation of ACCO and downregulation of LRR receptor-like serine/threonine-protein kinase that mediates 1-aminocyclopropane-1-carboxylic acid (ACC) synthase [48] due to ethylene treatment for ripening of kiwifruit. The gene that encodes methyltransferase, an enzyme that functions as a barrier to prevent senescence [49] was also downregulated due to ethylene treatment. The above results imply that exogenous ethylene application could stimulate more ethylene biosynthesis which in turn causes a rise in respiration rate, biochemical changes, color changes and softening ( Figures 5-7). Besides, ethylene-related gene families such as ethylene-responsive transcription factor RAP2-10, ethylene-responsive transcription factor RAP2-3 and ethylene-responsive transcription factor 4 were upregulated, which consequently lead to higher respiration rate and faster ripening [18]. Moreover, downregulation of zinc finger protein-like expression could cause accelerated ripening [50].
However, the gene that encodes S-adenosylmethionine decarboxylase (SAMDC) was also upregulated due to exogenous ethylene treatment. The biosynthesis of polyamines, i.e., spermidine and spermine, are catalyzed by SAMDC [51]. Polyamines are effective inhibitors of ethylene synthesis during the early stages of fruit ripening by inhibiting the conversion of methionine and ACC to ethylene [52]. The result implies that ethylene-treated kiwifruit could respond to the exogenous stimuli by upregulation of the gene that encodes SAMDC immediately after treatment to inhibit further ethylene synthesis. Additionally, ethylene treatment caused the downregulation of ethylene-responsive transcription factor TINY-like in kiwifruit (Table 5). This could be due to its role before harvest in plant growth and development in the enhancement of a 'tiny' phenotype rather than its action after harvest [53]. Generally, ethylene affects ripening through the regulation of ethylene receptors and triggering signal transduction reactions which ultimately control relevant gene expression in the fruit [20].
Ethylene bursts during ripening and causes the climacteric rise of respiration ( Figure 5), which could be a death signal that prepares fruit for seed dispersal through the breakdown of cellular components [47]. In this study, the genes that encoded glycolysis and TCA cycling enzymes, which mediate climacteric respiration, such as ribose-5-phosphate isomerase and pyruvate kinase, were upregulated in ethylene-treated fruit. The results imply that the response of the enzymes after ethylene treatment could lead to faster ripening as compared with the control. The gene that encodes 6-phosphogluconolactonase, the protein related to fruit respiratory pathway and quality [54], was also upregulated due to ethylene treatment. This could be explained by the respiration burst caused by the higher ethylene concentration.

TSS, TA, BAR and Sensory Evaluation and the Related Genes
TSS, TA, BAR and the sensory evaluation in the present study reveal that ethylene-treated fruit was preferred over the control on the third day of ripening ( Figure 6). Phosphoenolpyruvate carboxylase (PEPC) is involved in organic acid synthesis [55]. The gene that encodes PEPC was downregulated after ethylene treatment due to the reduction of acidity ( Figure 6) during ripening that might be associated with the conversion of organic acids into sugars and their derivatives or their utilization in respiration [25]. β-Amylase (BAM), one of the key genes related to starch degradation during the ripening of fruit [18], was also upregulated in our study. BAM breaks starch into maltose, resulting in the sweet flavor of ripe fruit [56]. Furthermore, the upregulation of the sucrose-phosphate synthase (SPS) encoding gene could be related to an increase in TSS, BAR and sensory quality ( Figure 6). Similarly, Duque et al. [57] reported the accumulation of soluble sugars coincided with a gradual activation of SPS after harvest during cold storage. Besides, glucose-1-phosphate adenylyltransferase large subunit 2/amyloplastic-like protein, which plays a role in the synthesis of starch was downregulated, implying that fruit ripening involves catabolic processes rather than anabolic processes.
Methanol O-anthraniloyltransferase is an enzyme responsible for the production of O-methyl anthranilate, an important aroma and flavor compound in the grapefruit [58]. Similarly, methanol O-anthraniloyltransferase gene was upregulated due to ethylene treatment in this study, implying that O-methyl anthranilate could be produced during ripening, which can directly influence the aroma and flavor of 'ready to eat' kiwifruit. Hence, the methanol O-anthraniloyltransferase gene could be responsible for the development of aroma and flavor of kiwifruit during ripening. In this study, the gene that encodes arogenate dehydrogenase, an enzyme that catalyzes the oxidative decarboxylation of arogenate into the aromatic amino acid tyrosine [59], was also upregulated due to exogenous ethylene treatment, indicating that kiwifruit ripening is also associated with changes in aroma of the fruit. Interestingly, the genes that encode linoleate 13S-lipoxygenase and linoleate 9S-lipoxygenase, lipoxygenases that may contribute to aldehyde generation to produce grassy aroma during kiwifruit ripening [60], were downregulated due to ethylene treatment. The results of overall sensory evaluation and BAR in the current study ( Figure 6) could also be related to the above changes. However, 4-coumarate-CoA ligase-like, an enzyme that involved in phenylpropanoid-derived compound (PDC) pathway [61], carboxylesterase (CXE) which regulates plant bioactive metabolism through the control of esterases to release volatile carboxyl esters [62], UDP-glycosyltransferase which catalyzes the production of glycosides (a potential source of aroma and flavor in fruits) [63] and α-glucosidase, the key enzyme that catalyzes the final step in the digestive process of carbohydrates [64], were downregulated in the present study after 3 days of ethylene treatment, probably due to early action of these enzymes at the onset of ethylene treatment and reduced action afterward.
L-Ascorbate oxidase gene was upregulated due to ethylene treatment. L-ascorbic acid (AA), one of the major antioxidants in fruits and vegetables, known as vitamin C by most consumers, is oxidized to L-dehydroascorbic acid (DHAA), mainly due to the activity of L-ascorbate oxidase [65]. The biological activity of DHAA has been considered to be equivalent to AA as it can be converted readily into AA in the human body [66]. Besides, the enzyme that catalyzes the terminal step in the biosynthetic pathway of AA [67] could be enhanced due to the upregulation of L-gulonolactone oxidase (Table 4). Moreover, the other ascorbate-related genes in kiwifruit, including galacturonosyltransferase and UDP-glucose dehydrogenase, were upregulated due to ethylene treatment.
Agronomy 2019, 9, x FOR PEER REVIEW 2 of 18 downregulated, implying that fruit ripening involves catabolic processes rather than anabolic processes.
Methanol O-anthraniloyltransferase is an enzyme responsible for the production of O-methyl anthranilate, an important aroma and flavor compound in the grapefruit [58]. Similarly, methanol Oanthraniloyltransferase gene was upregulated due to ethylene treatment in this study, implying that O-methyl anthranilate could be produced during ripening, which can directly influence the aroma and flavor of 'ready to eat' kiwifruit. Hence, the methanol O-anthraniloyltransferase gene could be responsible for the development of aroma and flavor of kiwifruit during ripening. In this study, the gene that encodes arogenate dehydrogenase, an enzyme that catalyzes the oxidative decarboxylation of arogenate into the aromatic amino acid tyrosine [59], was also upregulated due to exogenous ethylene treatment, indicating that kiwifruit ripening is also associated with changes in aroma of the fruit. Interestingly, the genes that encode linoleate 13S-lipoxygenase and linoleate 9S-lipoxygenase, lipoxygenases that may contribute to aldehyde generation to produce grassy aroma during kiwifruit ripening [60], were downregulated due to ethylene treatment. The results of overall sensory evaluation and BAR in the current study ( Figure 6) could also be related to the above changes. However, 4-coumarate-CoA ligase-like, an enzyme that involved in phenylpropanoid-derived compound (PDC) pathway [61], carboxylesterase (CXE) which regulates plant bioactive metabolism through the control of esterases to release volatile carboxyl esters [62], UDP-glycosyltransferase which catalyzes the production of glycosides (a potential source of aroma and flavor in fruits) [63] and αglucosidase, the key enzyme that catalyzes the final step in the digestive process of carbohydrates [64], were downregulated in the present study after 3 days of ethylene treatment, probably due to early action of these enzymes at the onset of ethylene treatment and reduced action afterward.
L-Ascorbate oxidase gene was upregulated due to ethylene treatment. L-ascorbic acid (AA), one of the major antioxidants in fruits and vegetables, known as vitamin C by most consumers, is oxidized to L-dehydroascorbic acid (DHAA), mainly due to the activity of L-ascorbate oxidase [65]. The biological activity of DHAA has been considered to be equivalent to AA as it can be converted readily into AA in the human body [66]. Besides, the enzyme that catalyzes the terminal step in the biosynthetic pathway of AA [67] could be enhanced due to the upregulation of L-gulonolactone oxidase (Table 4). Moreover, the other ascorbate-related genes in kiwifruit, including galacturonosyltransferase and UDP-glucose dehydrogenase, were upregulated due to ethylene treatment.

Stress-Related Genes Due to Ethylene Treatment
Ethylene treatment also caused the upregulation of transcription factor MYB1R1 (Table 4). Shin et al. [68] reported that the expression of transcription factor MYB1R1 in potato plants improved plant tolerance to drought stress via regulation of water loss. Peptide methionine sulfoxide reductase and universal stress protein A-like protein genes that could be involved in the protective roles were also upregulated. In line with our results, Lopez et al. [69] and Tkaczuk et al. [70] reported the upregulation of peptide methionine sulfoxide reductase and universal stress protein A-like protein genes to

Stress-Related Genes Due to Ethylene Treatment
Ethylene treatment also caused the upregulation of transcription factor MYB1R1 (Table 4). Shin et al. [68] reported that the expression of transcription factor MYB1R1 in potato plants improved plant tolerance to drought stress via regulation of water loss. Peptide methionine sulfoxide reductase and universal stress protein A-like protein genes that could be involved in the protective roles were also upregulated. In line with our results, Lopez et al. [69] and Tkaczuk et al. [70] reported the upregulation of peptide methionine sulfoxide reductase and universal stress protein A-like protein genes to overcome stress conditions. Moreover, there was an upregulation of the gene that encodes peroxidase, the enzyme that catalyzes the reduction of peroxides and the polymerization of toxic compounds [71]. Hence, the upregulation of transcription factor MYB1R1, peptide methionine sulfoxide reductase, universal stress protein A-like protein genes and peroxidase in the present study could be the response of the fruit to the stress caused by the exogenous ethylene treatment.
Phenylalanine ammonia-lyase (PAL), one of the key enzymes in the phenylpropanoid pathway [72], and 3-ketoacyl-CoA synthase (KCS) were also upregulated due to ethylene treatment ( Table 4). The phenylpropanoid pathway produces phenolic compounds that contribute to fruit pigmentation and the disease resistance response during the ripening process of fruits [73]. Han et al. [74] reported increased transcript levels of the KCS gene, a gene that involves very long-chain fatty acid biosynthesis of lipid pathway following the application of abscisic acid (ABA) for wound suberization. Furthermore, there was an upregulation of the gene that encodes β-carotene hydroxylase, an enzyme in the zeaxanthin biosynthetic pathway which could help for protection against stress [75]. Therefore, ethylene treatment could intensify the phenylpropanoid metabolism, lipid metabolism and zeaxanthin biosynthesis through activating PAL, KCS and beta-carotene hydroxylase enzymes during kiwifruit ripening.
In contrast to Park et al. [18] who reported the downregulation of polyphenol oxidases (PPOs) after treatment of persimmon fruit, treatment of kiwifruit with ethylene in the present study upregulated PPOs. PPOs are involved in production of brown pigments in wounded tissues by catalyzing the oxidation of phenolic compounds to quinones [76]. In this study, the treatment of kiwifruit with exogenous ethylene hastens the softening as compared with control ( Figures 5 and 7), and the fruit could be liable to physical damage which may result in wounded tissues. Besides, superoxide dismutase (SOD), the first line of defense against oxidative stress [77], subtilisin-like protease which is associated with pathogen resistance and plant immunity [78], ABC transporter G family member 3-like which is mainly involved in detoxification process and response to abiotic stress [79] and receptor-like protein kinase (RLK), the transmembrane proteins that perceive the signals of environmental conditions [80], were downregulated. Hence, care should be taken not to ripen the kiwifruit over three days after treatment with ethylene as it may lead to oversoftening that favors postharvest loss and browning.
The gene that encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an essential component of the glycolytic pathway which converts glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate was upregulated in this study. In addition to its key role in glycolysis, GAPDH is involved in abiotic stress resistance in plants [81]. The gene that encodes adenylate isopentenyltransferase, a cytokinin (CK) biosynthesis enzyme, was also upregulated with ripening due to ethylene treatment. In line with our results, [82] indicated the increasing levels of CK in green and gold kiwifruit during ripening. Ha et al. [83] reported that CK regulates responses to environmental stresses in plants. Besides, NAC transcription factor was upregulated in this study. Stress-inducible NAC genes are involved in abiotic stress response [84]. In this study, ethylene-induced ripening in kiwifruit also evokes the upregulation of the prolyl-4-hydroxylase gene, consistent with [85], who reported the expression of the same gene during ripening of banana, with it being involved in stress, defense and detoxification. Moreover, Chen et al. [86] suggested that WRKY transcription factors play important roles associated with stress responses in plants. Our results also showed upregulation of the WRKY transcription factor gene indicating that kiwifruit responds toward the stress caused by the exogenous application of ethylene. Zhu et al. [87] reported that exogenous niacin treatment during the growth of kiwifruit increases NADPH oxidase to increase tolerance to short-term stress conditions. In the present study, the upregulation of the gene that encodes NADPH oxidase after exogenous application of ethylene could be the response to the stress caused by ethylene treatment.

Color and Other Changes and the Related Genes
The genes that encode the light-harvesting complexes including chlorophyll a-b binding protein-like, chlorophyll a-b binding protein 3C precursor and chlorophyll a-b binding protein 37 precursor were downregulated due to ethylene treatment. Chlorophyll (ide) b reductase, which plays a central role in chlorophyll a-b binding protein degradation [88], and magnesium-protoporphyrin IX monomethyl ester (oxidative) cyclase, which has a great role in the process of chlorophyll biosynthesis [89], were also downregulated. Similarly, triose phosphate/phosphate translocator TPT-like that could catalyze the exchange of triose phosphate, 3-phosphoglycerate and inorganic phosphate across the chloroplast and plays crucial roles in photosynthesis [90] was downregulated. This could be attributed to the conversion of chloroplasts into chromoplasts, with the associated accumulation of colored carotenoids as fruits ripen [91] which causes color changes as observed in Figure 7. Additionally, the gene that encodes ribulose bisphosphate carboxylase/oxygenase (rubisco) activase, which plays a key role in photosynthesis [92], was downregulated.
In the present study, the amino-acid-metabolizing glutamate dehydrogenase B and aspartate aminotransferase encoding genes were also upregulated due to ethylene treatment. The coordinated events of the ripening process involve the changes at physiological and biochemical levels including the changes of chloroplasts into chromoplast which accompanied the metabolism of nitrogen compounds such as amino acids [93]. Similarly, Boggio et al. [93] also reported the upregulation of glutamate dehydrogenase B and aspartate aminotransferase in fully ripened red tomato fruits. However, the genes that encode 3-hydroxyisobutyrate dehydrogenase-like which is involved in branched-chain amino acid catabolism at carbohydrate limitation [94], (-)-isopiperitenol/(-)-carveol dehydrogenase which is involved in secondary metabolism of phenylpropanoids and terpenoids [95] and flavonoid 3'-monooxygenase(F3'H) which is involved in anthocyanin biosynthesis [96] were downregulated due to ethylene treatment. Downregulation of the genes encoding the above enzymes could imply the full ripening of kiwifruit before 3 days after treatment with exogenous ethylene.
In our results, we also found that gibberellin 2-β-dioxygenase was upregulated due to ethylene treatment. Gibberellin 2-β-dioxygenase catalyzes the 2-β-hydroxylation of biologically active gibberellins, which leads to the homeostatic regulation of their endogenous level. Similarly, Onik et al. [97] also reported the upregulation of gibberellin 2-β-dioxygenase during postripening of apple fruit, which could be related to the action of ethylene on ripening. Auxin regulates every aspect of plant growth and development by controlling gene expression through auxin response factors [98]. In the present study, the gene that encodes auxin response factor-like was downregulated due to ethylene treatment. From this result, it can be presumed that these two hormones may act antagonistically during the ripening of kiwifruit.
Agronomy 2019, 9, x FOR PEER REVIEW 4 of 18 monomethyl ester (oxidative) cyclase, which has a great role in the process of chlorophyll biosynthesis [89], were also downregulated. Similarly, triose phosphate/phosphate translocator TPTlike that could catalyze the exchange of triose phosphate, 3-phosphoglycerate and inorganic phosphate across the chloroplast and plays crucial roles in photosynthesis [90] was downregulated. This could be attributed to the conversion of chloroplasts into chromoplasts, with the associated accumulation of colored carotenoids as fruits ripen [91] which causes color changes as observed in Figure 7. Additionally, the gene that encodes ribulose bisphosphate carboxylase/oxygenase (rubisco) activase, which plays a key role in photosynthesis [92], was downregulated.
In the present study, the amino-acid-metabolizing glutamate dehydrogenase B and aspartate aminotransferase encoding genes were also upregulated due to ethylene treatment. The coordinated events of the ripening process involve the changes at physiological and biochemical levels including the changes of chloroplasts into chromoplast which accompanied the metabolism of nitrogen compounds such as amino acids [93]. Similarly, Boggio et al. [93] also reported the upregulation of glutamate dehydrogenase B and aspartate aminotransferase in fully ripened red tomato fruits. However, the genes that encode 3-hydroxyisobutyrate dehydrogenase-like which is involved in branched-chain amino acid catabolism at carbohydrate limitation [94], (-)-isopiperitenol/(-)-carveol dehydrogenase which is involved in secondary metabolism of phenylpropanoids and terpenoids [95] and flavonoid 3'monooxygenase(F3'H) which is involved in anthocyanin biosynthesis [96] were downregulated due to ethylene treatment. Downregulation of the genes encoding the above enzymes could imply the full ripening of kiwifruit before 3 days after treatment with exogenous ethylene.
In our results, we also found that gibberellin 2-β-dioxygenase was upregulated due to ethylene treatment. Gibberellin 2-β-dioxygenase catalyzes the 2-β-hydroxylation of biologically active gibberellins, which leads to the homeostatic regulation of their endogenous level. Similarly, Onik et al. [97] also reported the upregulation of gibberellin 2-β-dioxygenase during postripening of apple fruit, which could be related to the action of ethylene on ripening. Auxin regulates every aspect of plant growth and development by controlling gene expression through auxin response factors [98]. In the present study, the gene that encodes auxin response factor-like was downregulated due to ethylene treatment. From this result, it can be presumed that these two hormones may act antagonistically during the ripening of kiwifruit.

Conclusions
This work presents the transcriptome sequencing analysis of 'Haegeum' gold kiwifruit after treatment with ethylene. Several genes were expressed differentially during the ripening of kiwifruit, and the cumulative effect brings the softening-and ripening-related changes. The results indicated that there is no single gene or enzyme that accounts for kiwifruit ripening, rather, there are many potentially responsible genes that exhibit ripening-related expressions. We identified and categorized genes related to softening and other changes during ripening. The data obtained from the present study will add to the information available on the molecular mechanisms of the effects of ethylene during ripening of kiwifruit. This study will also provide important resources for further study of the genes related to ripening for kiwifruit breeding and improvement.

Conclusions
This work presents the transcriptome sequencing analysis of 'Haegeum' gold kiwifruit after treatment with ethylene. Several genes were expressed differentially during the ripening of kiwifruit, and the cumulative effect brings the softening-and ripening-related changes. The results indicated that there is no single gene or enzyme that accounts for kiwifruit ripening, rather, there are many potentially responsible genes that exhibit ripening-related expressions. We identified and categorized genes related to softening and other changes during ripening. The data obtained from the present study will add to the information available on the molecular mechanisms of the effects of ethylene during ripening of kiwifruit. This study will also provide important resources for further study of the genes related to ripening for kiwifruit breeding and improvement.