Journal of Applied Botany and Food Quality 92, 138 - 142 (2019), DOI:10.5073/JABFQ.2019.092.019 1Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang, Hebei, PR China 2Hebei University of Environmental Engineering, Qinhuangdao, Hebei, PR China 3Plant Genetic Engineering Center of Hebei Province, Shijiazhuang, PR China 4Hebei Normal University of Science & Technology, Qinhuangdao, Hebei, PR China Different response to 1-methylcyclopropene in two cultivars of Chinese pear fruit with contrasting softening characteristics Jianmei Wei1, 2 a, Yudou Cheng 1, 3 a, Yunxiao Feng1, 3, Xiudong Qi 4, Jingang He1, 3, Junfeng Guan1, 3 * (Submitted: January 9, 2019; Accepted: February 14, 2019) * Corresponding author a These authors contributed equally to the article Summary In this study, the change in softening and its related genes expres- sion under influence of 500 nl L-1 1-methylcyclopropene (1-MCP) was assessed in the two Chinese pear fruit, ‘Jingbaili’ (Pyrus ussuriensis Maxim) and ‘Yali’ (Pyrus bretschneideri Rehd), which exhibit dif- ferent softening characteristics. ‘Jingbaili’ pear fruit softened rapidly after harvest, and was strongly inhibited by 1-MCP. In contrast, there was no obvious change of firmness compared to the control after 1-MCP treatment in ‘Yali’ pear fruit. The respiration and ethylene production rates were reduced by 1-MCP at early storage in both two cultivars. ‘Jingbaili’ pear fruit exhibited dramatically increased expression levels of the softening-related genes, i.e., polygalacturo- nase1 (PG1), polygalacturonase2 (PG2), β-Galactosidase4 (GAL4), α-arabinofuranosidase1 (ARF1) and α-arabinofuranosidase2 (ARF2), and these genes’ expression levels were significantly decreased by 1-MCP treatment. In contrast, ‘Yali’ pear fruit showed lower ex- pression levels of the above-mentioned genes, as well as a relatively smaller inhibition effect by 1-MCP treatment before day 27. These results suggest that ‘Jingbaili’ pear fruit are more sensitive to 1-MCP/ ethylene than ‘Yali’ pear fruit during ripening. Keywords: pear; 1-methylcyclopropene; softening; cell wall en- zyme; gene expression Introduction ‘Jingbaili’ (Pyrus ussuriensis Maxim) and ‘Yali’ (Pyrus bretschnei- deri Rehd) pear fruit are famous cultivars and belong to typical Chinese pear with different softening and ripening behaviors. ‘Jing- baili’ pear fruit exhibits an extremely rapid decrease in flesh firmness during ripening, while ‘Yali’ pear fruit do not exhibit fruit softening and maintain a crispy texture even during the late stage of ripening (Hiwasa et al., 2004; Wei et al., 2009, 2015). 1-methylcyclopropene (1-MCP), an effective inhibitor of ethylene action, has been found to delay softening of fruits, such as apple, banana, kiwifruit, mango, tomato and durian by blocking the ethy- lene signal transduction pathway, and has been widely used to inves- tigate fruit tissue responses to ethylene during ripening and senes- cence of climacteric fruit (Golding et al., 1998; Jiang and Joyce, 2002; Gamrasni et al., 2010; Vilas-Boas et al., 2007; Piriyavinit et al., 2011; Lu et al., 2012; Amornputti et al., 2014). Cell wall disassembly is believed to contribute to fruit softening (Wakabayashi, 2000; Sañudo-Barajas et al., 2009). The depoly- merization and solubilization of the pectin polymer is the major fac- tor involved in fruit softening, and the pectin modification that cause cell dispersion and hydrolysis of the cell wall polymers is associated with texture change (Brummell, 2006; Goulao et al., 2008; Gwan- pua et al., 2014). Many studies have shown that pectin modification is caused by the action of a series of cell wall-modifying enzymes and proteins, such as polygalacturonase (PG), β-Galactosidase (β-GAL) and α-arabinofuranosidase (α-ARF) (Brummell, 2006; Gwanpua et al., 2014; Wei et al., 2015). These enzymes have the capacity to reduce intercellular connections and the molecular size of pectin polymers by cleaving the backbone or the side chain residues. The modified polymers play different roles in fruit softening (Sañudo- Barajas et al., 2009; Wei et al., 2015). In this study, we investigated the effects of 1-MCP on fruit softening of ‘Jingbaili’ and ‘Yali’ pears. Furthermore, we measured changes in the expression patterns of the softening-related genes (PG1, PG2, β-GAL4, ARF1 and ARF2) to analyze the differences in the respon- ses to 1-MCP between two cultivars of pear fruits. Materials and methods Biological materials and treatments ‘Jingbaili’ (Pyrus ussuriensis Maxin.) and ‘Yali’ (Pyrus bretschnei- deri Rehd.) pear fruit were harvested at commercial maturity (For ‘Jingbaili’ pear fruit: Average weight: 101.7 ± 5.8 g, TSS: 12.2 ± 0.9%; For ‘Yali’ pear fruit: Average weight: 179.2 ± 10.7 g, TSS: 10.9 ± 1.0%) (Sep. 15, 2014) from an orchard in Changli County, Hebei Province, China. The fruit without mechanical injury, insects and diseases were placed directly in a sealed container with 500 nL L-1 1-MCP (0.18%, Ansip®, Taiwan, China), After fumigating for 24 h at 20 ± 1 °C, the fruit were then ventilated and stored at 20 ± 1 °C. Control fruit were subjected to the same protocol with the excep- tion of a lack of 1-MCP treatment in the sealed container. Fruit were sampled at appropriate intervals based on their softening rates, the flesh tissues were frozen in liquid nitrogen and stored at -70 °C for subsequent analysis. Determination of fruit firmness, respiration rate, ethylene pro- duction rate Flesh firmness was determined using a digital fruit penetrometer (TuoPu Instruments, Model GY-4, Zhejiang, China) with ten fruit per sampling time, and three replications per treatment. The respira- tion and ethylene production rates were measured via the air-stream method with three replications per treatment. Ethylene production rate was determined by using a gas chromatograph (Lunan Ruihong Chemical Instruments, Model GC-SP-6890, Shandong, China). 2.0 kg of fruit were placed in a 9.5 L airtight jar for 1 h at 20 ± 1 °C. The 60 μL of gas samples were withdrawn through a septum on top of the jar using a gas-tight syringe, and three replications per treatment. RNA extraction and qRT-PCR analysis Total RNA was extracted from the pear fruit samples using the modified CTAB method (gasic et al., 2004). Based on our previ- ous research (Wei et al., 2015), we choose PG1, PG2, GAL4, ARF1 Two Chinese pear response to 1-MCP 139 and ARF2 to be measured by quantitative reverse transcription- polymerase chain reaction (qRT-PCR). First-strand cDNAs were synthesized from DNase-digested RNA (0.5 μg) using the Takara RNA PCR Kit (AMV) Version 3.0 (TaKaRa Biomedicals, Japan). qRT-PCR was performed using the SYBR Premix Ex TaqTM (Perfect Real Time) Kit (TaKaRa Biomedicals, Japan) on a 7500 Real-Time PCR system (Applied Biosystems, USA). qRT-PCR primers for PGs, GAL4 and ARFs have been reported by Wei et al. (2015). The qRT- PCR reaction was performed in a final volume of 25 μL containing 12.5 μL SYBR Green PCR Premix Ex TaqTM, 1 μmol L-1 forward and reverse primers, and 10 ng cDNA with cycles as follows: 10 s at 95 °C, 40 cycles of 95 °C for 5 s, and 60 °C for 34 s. The Tm of the amplification products was analyzed using a dissociation curve to confirm the quality of the PCR product and primer specificity. Actin2 (ACT2) was used as a reference gene. All qRT-PCR reactions were normalized using Ct value corresponding to the Actin2 gene. The relative expression levels of the detected genes were calculated with the formula 2 -ΔΔCT and the experiment was performed with four replicates per treatment. Statistical analysis The results were subjected to ANOVA statistically analyzed using SPSS 18 software (SPSS Inc., Chicago, IL, USA). All of the values are expressed as the means ± SD of three replicates. Least significant differences (LSD) at a significance level of 0.05 were generated by ANOVA. Results Respiration and ethylene production rates and flesh firmness In ‘Jingbaili’ pear fruit, the respiration and ethylene production rate reached their peaks at the day 9 of storage concomitant with a rapid decrease in flesh firmness during ripening. After exposure to 1-MCP, the respiration and ethylene production rates dramatically decreased at first. And moreover, their climatic peaks were all delayed, and flesh firmness was noticeably kept at an initial value (Fig. 1 A, C and E). In contrast, although 1-MCP resulted in a decrease trend in the respiration during storage, as well as the ethylene production rates before day 27 of storage in ‘Yali’ pear, it did not exhibit the marked loss of flesh firmness (Fig. 1 B, D and F). PG gene expression Both the PG1 and PG2 transcripts were expressed at very high levels and were rapidly enhanced before day 12 of storage in ‘Jingbaili’ pear fruit, and both of the PG1 and PG2 mRNA accumulated slowly and were dramatically reduced in 1-MCP treatment than in control, except at day 15 for PG2 (Fig. 2A and C). Relatively, the mRNA amounts of PG1 and PG2 were much lower in ‘Yali’ pear than that in ‘Jingbaili’ pear, and the peaks were found at day 36 for PG1and day 18 for PG2. In addition, PG1 and PG2 were significantly decreased by 1-MCP at day 18 and day 27, respectively (Fig. 2 B and D). β-GAL gene expression The GAL4 is the major softening-related gene involved in fruit ripe- ning, consistent with the changes in β-GAL activity in pear fruit (wei et al., 2015). Therefore, the expression level of GAL4 was stu- died here. The mRNA of GAL4 accumulated rapidly and was dra- matically inhibited by 1-MCP in ‘Jingbaili’ pear before day 12 of storage (Fig. 3A). The expression of GAL4 in ‘Yali’ pear rose to a peak at day 18 in control, meanwhile, it increased slowly before day 27 of storage and afterwards reached the peak at day 36 in 1-MCP treatment (Fig. 3B). Fig. 1: Effects of 1-MCP on the rates of respiration (A and B) and ethylene production (C and D), and firmness (E and F) in ‘Jingbaili’ (A, C and E) and ‘Yali’ (B, D and F) pear fruit. Values are the means ± SD. Fig.2: Effect of 1-MCP on the expression of PGs in ‘Jingbaili’ (A and C) and ‘Yali’ (B and D) pear fruit. Values are the means ± SD. 140 J. Wei, Y. Cheng, Y. Feng, X. Qi, J. He, J. Guan α-ARF gene expression The expression level of ARF2 in ‘Jingbaili’ pear was higher and in- creased more rapid than that of ARF1, while the expression levels of the ARF1 and ARF2 genes increased slowly and showed much lower levels in 1-MCP treatment than that in control (Fig. 4A and C). However, both ARF1 and ARF2 expression were lower and there was a lower level of ARF1 expression at day 9 and 18, and ARF2 at day 9, 18 and 27, even though there was a higher expression level of ARF1 in 1-MCP treatment during the later stage in ‘Yali’ pear fruit (Fig. 4B and D). et al., 2009; minas et al., 2013), mangosteen (piriyavinit et al., 2011), apple (yang et al., 2013; Ireland et al., 2014), pear (Xie et al., 2014; Escribano et al., 2017), tomato (Dong et al., 2013) and musk- melon (Supapvanich et al., 2013). In this study, application of 1-MCP after harvest dramatically delayed softening in ‘Jingbaili’ pear fruit but had little effect in ‘Yali’ pear fruit (Fig. 1E and F), suggesting that ‘Jingbaili’ pear is more sensitive to ethylene for regulating fruit softening than ‘Yali’ pear. An increase in PG activity and mRNA level has been reported in several fruit species concomitant with the degradation of pectin polysaccharides and softening (Hiwasa et al., 2003, 2004; Wei et al., 2015, Song et al., 2016; Gwanpua et al., 2016). 1-MCP treatment restricted softening and reduced PG activity and the expression level of PGs genes in pear and papaya fruits after the onset of ripening (Hiwasa et al., 2003, 2004; Zerpa-Catanho et al., 2017). A similar result was observed in the ‘Jingbaili’ pear fruit, which both PG1 and PG2 expression presented at much higher levels in control and were sharply suppressed by 1-MCP during the onset of softening in the ‘Jingbaili’ pear fruit (Fig. 2A and C). By contrast, PGs expression in ‘Yali’ pear fruit was relatively less affected by 1-MCP treatment (Fig. 2B and D). Ranwala et al. (1992) found that purified β-GAL had the capa- city to catalyze an apparent decrease in the molecular size of pec- tins in vitro. In strawberry, down-regulated of Faβ-GAL4 increases cell wall galactose levels and reduces fruit softening (Paniagua et al., 2016). 1-MCP treatment inhibited mRNA accumulation of CmGAL in melon, suggesting that the expression of CmGAL is likely to be dependent on ethylene and may therefore function as a potential co-operative action partner with PG1 (Nishiyama et al., 2007). In this study, we found that the expression of GAL4 was dramatically inhibited by 1-MCP treatment in ‘Jingbaili’ pear fruit (Fig. 3A). In agreement with report that the expression of GAL4 was negatively correlated with water- and Na2CO3-soluble polymers (rich in neutral sugars) in the ‘Jingbaili’ pear fruit (Wei et al., 2009). In contrast, the expression level of GAL4 was maintained at a low level under 1-MCP treatment in ‘Yali’ pear fruit with a slower increase at first and then dropped (Fig. 3 B). It was in agreement with the results reported by Mwaniki et al. (2005) and Tateishi et al. (2005). The possible involvement of a glycoside hydrolase such as α-ARF in cell wall modification during fruit ripening has been well docu- mented. ARF expression could result in the release of in many fruits (Sozzi et al., 2002; Brummell et al., 2004; Nishiyama et al., 2007; Wei et al., 2010). In ‘Jingbaili’ pear fruit, ARFs gene expression rapidly increased and were significantly inhibited by 1-MCP treat- ment (Fig. 4 A and C), while in ‘Yali’ pear fruit, they were main- tained at a lower level and exhibited only a slight inhibition by 1-MCP (Fig. 4 B and D). These results suggested that ARFs in ‘Jingbaili’ pear fruit were more sensitive to ethylene than those in ‘Yali’ pear fruit. Furthermore, α-ARF may play different roles in pear fruit softening due to the different textural attributes and response to ethylene as shown in tomato (Sozzi et al., 2002) and apple (Goulao et al., 2007). Conclusion In summary, this study indicated that the fruit softening was sig- nificantly inhibited by 1-MCP treatment in ‘Jingbaili’ pear, while no obvious change was observed in ‘Yali’ pear. This was caused by the different change of expression of the softening-related genes, i.e., PG1, PG2, GAL4, ARF1 and ARF2. This suggests that the ‘Jingbali’ pear is more sensitive to 1-MCP/ethylene than the ‘Yali’ pear during ripening. Acknowledgements This work was supported by the emarked fund for the China Ag- riculture Research System for National Technology System for Fig. 3: Effect of 1-MCP on the expression of GAL4 in ‘Jingbaili’ (A) and ‘Yali’ (B) pear fruit. Values are the means ± SD. Fig. 4: Effect of 1-MCP on the expression of ARFs in ‘Jingbaili’ (A and C) and ‘Yali’ (B and D) pear fruit. Values are the means ± SD. 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