The Role of 1‐Methylcyclopropene in the regulation of ethylene biosynthesis and ethylene receptor gene expression in Mangifera indica L. (Mango Fruit)

Abstract Mango (Mangifera indica L.) is respiratory climacteric fruit that ripens and decomposes quickly following their harvest. 1‐methylcyclopropene (1‐MCP) is known to affect the ripening of fruit, delaying the decay of mango stored under ambient conditions. The objective of this study was to clarify the role of 1‐MCP in the regulation of ethylene biosynthesis and ethylene receptor gene expression in mango. 1‐MCP significantly inhibited the 1‐aminocyclopropane‐1‐carboxylic acid (ACC) content. The activity of ACC oxidase (ACO) increased on days 6, 8, and 10 of storage, whereas delayed ACC synthase (ACS) activity increased after day 4. The two homologous ethylene receptor genes, ETR1 and ERS1 (i.e., MiETR1 and MiERS1), were obtained and deposited in GenBank® (National Center for Biotechnology Information‐National Institutes of Health [NCBI‐NIH]) (KY002681 and KY002682). The MiETR1 coding sequence was 2,220 bp and encoded 739 amino acids (aa). The MiERS1 coding sequence was 1,890 bp and encoded 629 aa, similar to ERS1 in other fruit. The tertiary structures of MiETR1 and MiERS1 were also predicted. MiERS1 lacks a receiver domain and shares a low homology with MiETR1 (44%). The expression of MiETR1 and MiERS1 mRNA was upregulated as the storage duration extended and reached the peak expression on day 6. Treatment with 1‐MCP significantly reduced the expression of MiETR1 on days 4, 6, and 10 and inhibited the expression of MiETR1 on days 2, 4, 6, and 10. These results indicated that MiETR1 and MiERS1 had important functions in ethylene signal transduction. Treatment with 1‐MCP might effectively prevent the biosynthesis of ethylene, as well as ethylene‐induced ripening and senescence. This study presents an innovative method for prolonging the storage life of mango after their harvest through the regulation of MiETR1 and MiERS1 expression.


| INTRODUC TI ON
Ethylene is a gaseous plant hormone that is involved in plant growth and development, including fruit ripening, fruit abscission, leaf senescence, seed germination, and organogenesis (Bleecker & Kende, 2000;Trujillo-Moya & Gisbert, 2012). It also regulates various stress responses in plants, including water deficits, mechanical wounds, and pathogen attacks (Jiang & Fu, 2000;Martínez, Gómez, & Gómez-Lim, 2001). Ethylene is generated following the catalysis of 1-aminocyclopropane-1-carboxylic acid (ACC) by an enzyme called ACC oxidase (ACO). Pathak, Asif, Dhawan, Srivastava, and Nath (2003) reported that ACO was constitutively expressed at low levels and was induced during banana fruit ripening. In addition, the transcriptional activity of the ACC synthase (ACS) gene may regulate the synthesis of ethylene.
The ethylene receptor is an upstream element that is encoded by a multigene family, playing a negative regulatory role in the ethylene signal transduction pathway. The receptor proteins encoded by each gene have different structures and levels of expression.
Five ETR1-like genes, which are ETR1 (Chang, Kwok, Bleecker, & Meyerowitz, 1993), ERS1 (Hua, Chang, Sun, & Meyerowitz, 1995), ETR2 , EIN4, and ERS2 , are identified in Arabidopsis plants. Although ETR1 and ERS1 are ubiquitously expressed in Arabidopsis plants, they demonstrate distinct expression patterns in different tissues . The overall modular structures of ethylene receptors are similar, with a transmembrane domain near the N-terminus, accompanied by an undetermined functional GAF domain and domains for signal output in the C-terminus (Shakeel, Wang, Binder, & Schaller, 2013).
Three putative membrane-spanning subdomains were found in the N-terminal hydrophobic region of the ETR1 protein, which may assist in forming the ethylene-binding site. The C-terminus of ETR1 contains histidine protein kinase and receiving domains and may participate in delivering the ethylene signal. Although the protein encoded by the ERS1 gene has sequence similarities to the histidine kinase domain and the N-terminus of ETR1, it lacks a receiving domain. In a two-hybrid in vitro binding experiment, the C-terminal regions of both ETR1 and ERS1 were shown to interact with the rapidly accelerated fibrosarcoma kinase-like protein known as constitutive triple response 1 (CTR1) and to negatively regulate the transduction pathway of ethylene (Kuroda, Hirose, Shiraishi, Davies, & Abe, 2004).
It has been demonstrated that the ethylene action inhibitor 1-methylcyclopropene (1-MCP) affects plant senescence via irreversibly binding to the ethylene-binding receptor, thereby prohibiting the ethylene signal in the transduction pathway. Amornputti, Ketsa, and Doorn (2016) reported that 1-MCP inhibited the production of ethylene in fruit, which was correlated with the ACC content and the activity of ACO and ACS. Rasori, Ruperti, Bonghi, Tonutti, and Ramina (2002) reported that 1-MCP delayed peach fruit maturation by estimating the fruit firmness and release of ethylene. In addition, 1-MCP may downregulate ERS1 without affecting the transcription of ETR1 (Rasori et al., 2002). Li, Qiao, Tong, Zhou, and Zhang (2010) showed that 1-MCP upregulated the expression of the ETR3 gene in pear fruit 0 d-9 d after harvest and downregulated the expression of ERS2 6 d-15 d after harvest. Karakurt, Tonguç, and Ünlü (2014) confirmed that 1-MCP markedly decreased the expression levels of the ETR1, ERS1, and ETR2 genes in watermelons. Moreover, 1-MCP has been broadly used to delay ripening and senescence in plants and to consequently extend the storage life of climacteric fruit.
Mango is a fruit with a great market value. However, the storage life of mango is short (Sherman et al., 2015). Although mangos are harvested in the mature-green stage, they ripen quickly and often decay while being traded (Lalel, Singh, & Tan, 2003). The short postharvest life of mango limits their consumption (Salinas-Roca et al., 2018). Mango naturally produces ethylene; thus, controlling ethylene production is an appealing strategy to extend their storage life. Ascertaining the function of the ethylene receptors may assist in elucidating the regulatory mechanism of ethylene in postharvest mango. Unfortunately, the evidence on the expression of the ETR1 and ERS1 receptors in mango is currently limited. The shared common function and specific roles of ETR1 and ERS1 remain unknown. The role of 1-MCP in the regulation of ethylene receptor gene expression has not been investigated in mango. Therefore, it is necessary to elucidate the mechanisms of ethylene receptor gene expression in response to treatment with 1-MCP at the molecular level.
Regulation of ethylene biosynthesis has an impact on ripening and senescence of mango fruit. The aim of this study was to clarify the role of 1-MCP in the regulation of ethylene biosynthesis and ethylene receptor gene expression in mango after their harvest.
In particular, the role of 1-MCP in the regulation of MiETR1 and

| Plant materials and treatments
Experimental cultivar "Tainong 1" mango fruit (Mangifera indica L.) were harvested from an orchard in Tianyang, Baise City, Guangxi Province, China), in July 2016. Fruits with a similar shape, size, and physiological maturity were selected as experimental materials. These fruits were transported to the Guangxi Key Laboratory of Fruits and Vegetables Storage-Processing Technology (Nanning, China) immediately after harvest and were randomly classified into two groups (60 fruits per group). 1-MCP (Beijing PLM Biosciences Co.) was purchased in a powder form and dissolved in sterile distilled water at a final concentration of 1 μl/L. One group was placed in polyethylene bags (0.03 mm thick) containing 1 μl/L 1-MCP. The other group was treated with the same volume of sterile water only (control group). The fruits were treated with 1-MCP for 24 hr at 25°C and subsequently stored at 25°C and were then collected every two days, frozen in liquid nitrogen, and stored at −80°C until analysis.
Three replicates per analysis were used for all measurements.

| Evaluation of firmness, TSS,and TA of mango fruit
Firmness in the 1-MCP-treated and control groups was measured using a handheld FT-327 penetrometer (UC Fruit Firmness Tester, Milano, Italy) with an 8 mm diameter probe. Following the removal of a small piece of fruit skin, the firmness from three slices extracted from three different parts of the mango was recorded (means are presented in newtons [N]). The total soluble solid (TSS) content of the fruit was examined on the basis of the AOAC method (AOAC, 2000) using a handheld refractometer (ATAGO, Tokyo, Japan).
Mango fruit juice was titrated with 0.1 N NaOH to pH 8.2 in order to determine the titratable acidity (TA) using an automatic titrator (TitroLine Easy, Schott, Mainz, Germany).

| Rates of ethylene production and respiration
Postharvest fruits from the 1-MCP-treated and control groups were placed in pure N 2 and subsequently stored at 20°C with 90% relative humidity. Three fruits were placed in a 4.2 L airproof glass jar for 2 hr at 25°C to determine the rates of ethylene production at different storage stages. A headspace gas sample (1 ml) was collected from each jar and analyzed using a GC-2014C gas chromatograph (Shimadzu, Kyoto, Japan). Subsequently, a thermal conductivity detector (Shimadzu TCD-2014) with Porapak N column was used to detect the concentration of carbon dioxide in the samples. The levels of ethylene were determined using a flame ionization detector and an OV-17 capillary column (Zhonghuida Co.) (An, Zhang, Lu, & Zhang, 2006). The rates of ethylene production and respiration are presented on a FW basis that mention above.

| Determination of the ACC content and activity of the ACO and ACS
A total of 10 g of mango fruit pulp was collected from the 1-MCPtreated and control groups and homogenized in 25 ml of 80% (v/v) ethanol at 4°C. The homogenates were centrifuged for 10 min at 10,000g, and the supernatant ethanol was removed using a SpeedVac ™ concentrator (Thermo Fisher Scientific, Waltham). The remaining pellets were resuspended in distilled water, and the ACC content was determined on the basis of the method described by Boller, Herner, and Kende (1979) and the protocol by Lizada and Yang (1979). The extraction of ACO and ACS was conducted, and their activities were determined according to the method described by Zheng, Nakatsuka, Taira, and Itamura (2005).

| RNA extraction
Total RNA was isolated from 100 mg of fresh mango tissue pulverized using liquid nitrogen. RNA was isolated on the basis of the method described by López-Gómez and Gómez-Lim (1992). An RNAprep Pure Kit for plants (Tiangen Co., Beijing, China) was utilized according to the manufacturer's instructions, followed by treatment with RNase-free DNase (Takara Biotechnology) in order to purify the samples. The RNA quantity was assessed using a NanoDrop ® ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies Inc.) at 260 nm. A 2,100 Bioanalyzer system (Agilent Technologies) was used to evaluate RNA integrity.

| Full-length cDNA cloning of MiETR1 and MiERS1
Full-length cDNA sequences encoding MiETR1 and MiERS1 were acquired from the combination of RT-PCR and rapid amplification of cDNA ends (RACE) cloning. RNA (10 µg) was used to synthesize firststrand cDNA. All primers and reagents in cloning were purchased from the PowerScript ™ MMLV reverse transcriptase (Clontech). for 3 min; 94°C for 30 s, 72°C for 3 min following 5 cycles; 94°C for 30 s, 70°C for 3 min following five cycles; 94°C for 30 s, 68°C for 3 min following 25 cycles; and 68°C for 3 min. The products of 5′and 3′-RACE PCR were purified and cloned into a pMD18-T vector (Takara Bio) for sequencing (You et al., 2014). The full-length cDNA sequences of MiETR1 and MiERS1 were acquired according to the sequencing results of the conservative region and the 5′-and 3′-RACE products. which created from 1,000 replicates.

| Real-time quantitative PCR analysis
The cDNA of postharvest mango stored for various lengths of time was obtained as described earlier in this article. These cDNA samples were used to conduct a quantitative RT-PCR (RT-qPCR) assay.
The sequences designed for the MiETR1 gene-specific primers were  (Livak & Schmittgen, 2001). A seriated template dilution experiment was conducted using the quantization method, and a calibration curve was created. Agarose gel electrophoresis was used to determine the PCR products. The size of the expected amplicon was 130 bp.

| Statistical analysis
All experiments were performed in triplicate (n = 3) with a completely randomized design. All data are presented as mean ± standard error (SE). Fisher's least significant difference test was performed using one-way analysis of variance by SPSS software (version 13.0, IBM, New York, USA) with a significant value of p < .05.

F I G U R E 1
Effects of 1-MCP on firmness (a), TSS (b), and TA (c) of mango fruit at 25°C during sixteen days. Results represent the mean ± standard error (SE)

| Effects of treatment with 1-MCP on firmness, TSS, and TA of mango fruit
A decreasing trend in the firmness of mango was observed during ambient storage in both 1-MCP-treated and control groups ( Figure 1a). However, after four days of storage, loss of firmness was significantly delayed (p < .05) in the 1-MCP-treated group compared with that observed in the control group. The TSS content in the pulp tissues of both groups was shown to increase as the storage duration extended (Figure 1b). However, the TSS content in the 1-MCP-treated group was significantly lower than that recorded in the control group, especially on days 4, 6, and 14 of storage (all p < .05). The TA content in the pulp tissues of both groups decreased during the 16-day storage. However, the TA content in the 1-MCP-treated group decreased more slowly than that observed in the control group after four days of storage ( Figure 1c). These results revealed that treatment with 1-MCP is effective in retaining the firmness of mango stored under ambient conditions.

| Effects of treatment with 1-MCP on the rates of ethylene production, respiration, and biosynthesis
Ethylene is naturally produced in postharvest mango and quickly leads to overripening. Initially, the rates of ethylene production in both groups showed an increasing trend, followed by a slow decline. Treatment with 1-MCP significantly prevented the production of ethylene after two days of storage (p < .05). Production of ethylene in the 1-MCP-treated group peaked on day 10 of storage, two days later compared with the control group. These results indicated that treatment of fruit with 1-MCP after harvest may suppress the climacteric production of ethylene. An increased respiration rate may be associated with fruit senescence and the development of disease during fruit storage. In this study, the respiration rate of mango showed a typical climacteric pattern under ambient storage (Figure 2b). In the 1-MCP-treated group, the respiration rate was significantly lower than that recorded in the control group after six days of storage (p < .05). In the 1-MCP-treated group, peak respiration was reached more slowly than in the control group, indicating that treatment with 1-MCP reduced the respiration of mango.
In both groups, the ACC content was initially increased, followed by a decrease throughout the entire storage duration In the 1-MCP-treated group, the ACS activity was lower than that observed in the control group after four days of storage (p < .05).
These results confirmed that treatment with 1-MCP inhibited the production of ethylene. This effect was closely related to the ACC content and the activities of ACO and ACS.

| Cloning and sequences of the MiETR1 and MiERS1 genes
The full-length cDNAs of two ethylene receptor genes in mango were obtained (Figure 4).

| Bioinformatic analysis
A secondary structural analysis for MiETR1 and MiERS1 was carried out (http://www.expasy.org). The results revealed that the putative F I G U R E 2 Effects of 1-MCP treatment on ethylene production (a) and respiration rate (b) in mango fruit at 25°C during sixteen days. Results represent the mean ± standard error (SE) MiETR1 peptide was composed of 24.37% alpha helix, 9.97% beta turn, and 42.93% random coil. The MiERS1 peptide consisted of 43.45% alpha helix, 7.52% beta turn, and 29.94% random coil. The major part of the secondary structure of MiETR1 was random coil, whereas the alpha helix was the major segment of the MiERS1 secondary structure.
In addition, both the N-and C-terminal parts were alpha helices. The The MiERS1 and MiERS1 proteins, which contained three conserved regions (i.e., HisKA, ATPase_c, and GAF domains), belonged to the ETR1 subgroup ( Figure 4). All proteins conserved the following residues: Ala31, Ile62, Cys65, Ala102, Cys4, Cys6, His354, and Asp659. Among these, Ala31, Ile62, Cys65, and Ala102 are considered to play an important role in the ordinary function of ethylene receptors (Bleecker & Schaller, 1996), the Cys4 and Cys6 residues are necessary to form the disulfide-linked dimer (Schaller & Bleeker, 1995). Moreover, His354 and Asp659 were the presumptive sites of histidine kinase and autophosphorylation of receiver domains (Chang et al., 1993). MiERS1 and MiERS1 showed 44% homology, and the homology of the N-terminal amino acids was higher than that observed for the C-terminal amino acids.
Moreover, MiERS1 lacked 110 aa and a signal receptor domain at the C-terminal compared with MiERS1. The low identity observed between MiETR1 and MiERS1 may be primarily related to the lack of a receiver domain in MiERS1. The MiETR1 and MiERS1 proteins were conserved at residues deemed to be crucial for the normal function of ETR1.
A phylogenetic tree was generated using MiETR1 and MiERS1 sequences with other related ETRs and ERSs deposited in GenBank ® (NCBI-NIH) ( Figure 6). As shown in Figure 6, mango MiETR1 shared a homology with that of other fruit.

| Expression analysis of MiETR1 and MiERS1 treated with 1-MCP
The relationship between the climacteric production of ethylene and the levels of MiETR1 and MiERS1 mRNA in postharvest mango was further analyzed. Real-time PCR using gene-specific primers was used to explore the expression patterns of MiETR1 and MiERS1 genes. In both groups, the expression of MiETR1 mRNA was shown to be upregulated as the storage duration extended (Figure 7a). The expression peaked on day 6 (6.47-fold higher levels vs. those observed in the control group on day 0), followed by a decrease on day 8. A similar pattern was recognized in an ETR-type gene in muskmelon (Sato-Nara et al., 1999) and tomato (LeETR4 and LeETR5) (Tieman & Klee, 1999). Treatment of mango with 1-MCP was linked to a reduction in the expression of MiETR1. A relatively higher expression of  (Golding, Shearer, Wyllie, & McGlasson, 1998), avocado (Feng, Apelbaum, Sisler, & Goren, 2000), plum (Valero, Martinezromero, Valverde, Guillen, & Serrano, 2003), persimmon (Luo, 2007), pears (Lu, Cureatz, & Toivonen, 2009), and pineapples (Selvarajah, Bauchot, & John, 2001).Therefore, it is suggested that treatment of mango with 1-MCP may prolong the storage life and improve the storage quality.  (Rasori et al., 2002). In the present study, two ethylene receptor genes, namely MiETR1 and MiERS1, were extracted from mango and found to be similar to those identified in the corresponding genes of Citrus sinensis, Citrus clementina, Arabidopsis thaliana, and Glycine max (Figure 4). MiETR1 and MiERS1 are members of a multigene family found in numerous species (Bleecker, 1999). At the N-terminus, the clone also includes three hydrophobic regions, with features of ETR homologs ( Figure 5). The MiETR1 and MiERS1 genes contain a histidine kinase domain and a sensor domain, playing crucial roles in the normal function of ethylene receptors and ETR-or ERS-type proteins (Bleecker, 1999 Martínez et al., 2001). In this study, the mRNA levels of MiETR1 and MiERS1 increased dynamically during the early stages of mango fruit storage (Figure 7). The expression patterns of MiETR1 and MiERS1 in mango suggested that these two ethylene receptors hold important functions in ethylene signal transduction.
Furthermore, these findings indicated that MiETR1 and MiERS1 might be putative ethylene receptors with an ethylene-binding ability. Treatment with 1-MCP suppressed the expression of MiETR1 and MiERS1 in mango stored at room temperature. These findings indicated that the changes in the expression of ethylene receptor genes caused by 1-MCP treatment may contribute to the interaction between 1-MCP and ethylene receptors. Yamamoto et al. (1995) demonstrated that the genes coding for ACO and ACS are linked to the expression of the ethylene receptor in melon fruit. In mango, a similar ripening pattern was found, implying a link between the expression of the ethylene receptor and the activities of ACO and ACS (Pathak et al., 2003). The increased expression of the ethylene receptor, along with the higher sensitivity and production of ethylene, was discovered in petioles of tomato (Syariful et al., 2019). Inhibition of ethylene by 1-MCP is based on the ability of 1-MCP to irreversibly bind to ethylene receptors, consequently diminishing the normal increase in ACS and ACO enzyme activities during ripening and senescence (Binder & Bleecker, 2003).