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Ethylene biosynthesis is involved in regulating chilling tolerance and SlCBF1 gene expression in tomato fruit
Postharvest Biology and Technology 149 (2019) 139–147
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Ethylene biosynthesis is involved in regulating chilling tolerance and SlCBF1 gene expression in tomato fruit Wenqing Yua, Jiping Shengb, Ruirui Zhaoa, Qin Wangc, Peihua Mac, Lin Shena,
T
⁎
a
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China School of Agricultural Economics and Rural Development, Renmin University of China, Beijing, 100872, China c Department of Nutrition and Food Science, University of Maryland, College Park, MD20142, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ethylene biosynthesis SlACS2 Cold tolerance SlCBF1 Tomato fruit
Ethylene plays positive role in cold response of tomato fruit. CBFs (C-repeat/dehydration-responsive element binding factors) have been proved important in regulating plant chilling tolerance. Till now, the role that ethylene biosynthesis played in chilling response of tomato fruit is still unclear. In this study, we used antisense SlACS2 and wild-type Lichun tomato fruit as inhibited- and normal- ethylene biosynthesis models, in order to investigate whether inhibiting ethylene biosynthesis could affect chilling tolerance and SlCBF1 relative expression in tomato fruit. Results showed that antisense SlACS2 tomato fruit suffered more chilling injury, accompanied by higher malondialdehyde content and ion leakage, less proline and soluble protein content, as well as lower antioxidant enzymes activities, suggesting that inhibiting ethylene biosynthesis reduced tomato fruit chilling tolerance. Furthermore, SlCBF1 expression in transgenic fruit was lower than in WT, the second peak of SlCBF1 expression was coincidence with the peaks of both endogenous ethylene production and ACS activity, and there was significant correlation between ACC content and SlCBF1 relative expression, which implied that endogenous ethylene biosynthesis could contribute to upregulate SlCBF1 expression under cold stress. These results revealed that ethylene biosynthesis played important roles in regulating tomato fruit chilling tolerance and SlCBF1 relative expression in tomato fruit.
1. Introduction Low-temperature storage is a widely used technique to prolong the life and preserve quality of fruit, while storage of tropical and subtropical fruit at temperatures below 12 °C raises the risk of chilling injury (CI) (Cruz-Mendívil et al., 2015). Tomato (Solanum lycopersicum) is originated from subtropical, which is a typical cold-sensitive fruit that has been widely used as a model to investigate cold-induced responses (Constán-Aguilar et al., 2014; Zhao et al., 2011). ICE–CBF−COR is currently the most investigated cold stress signaling pathway, in which CBFs (C-repeat/dehydration-responsive element binding factors) bind to cold-induced genes, played a pivotal role in the modulation of cold response (Stockinger et al., 1997). Three CBF genes have been identified in tomato, in which only the SlCBF1 gene could be effectively induced by cold stress (Zhang et al., 2004).
Previous studies revealed that SlCBF1 expression level was positively correlated with cold tolerance in tomato fruit (Zhao et al., 2009a). Apart from cold stress, several endogenous signal molecules could also regulate SlCBF1 expression, such as nitric oxide, hydrogen peroxide, methyl jasmonate, salicylic acid, gibberellin, and ethylene (Zhao et al., 2011; Ding et al., 2015; Wang et al., 2017; Xinhua Zhang, 2012; Ding et al., 2016; Zhao et al., 2009b), which contribute to increase tomato plant/fruit cold tolerance in a CBF-dependent manner. Ethylene biosynthesis mainly occurs in the process of fruit ripening or suffering stress, the latter is also called stress-induced ethylene, which has been thought to play an important role in stress response including cold stress response (Barry et al., 2000). Previous study has documented that blocking ethylene action with 1-MCP resulted in decreased cold tolerance, while treatment with ethephon led to increased cold tolerance in tomato fruit (Zhao et al., 2009b). Moreover,
Abbreviations: CBF, C-repeat/dehydration-responsive element binding factor; SlERF2, solanum lycopersicum ethylene response factor 2; SAM, S-adenosyl-L-methionine; ACC, 1-aminocyclopropane-1-carboxylic acid; ACS, ACC synthase; AVG, 2aminoethoxyvinyl glycine; CI, chilling injury; MDA, malondialdehyde; APX, ascorbate peroxidase; CAT, catalase; SOD, superoxide dismutases; Ct, threshold cycle; qRT-PCR, quantitative real-time PCR; WT, wild-type; RT, room temperature; ROS, reactive oxygen species ⁎ Corresponding author. E-mail address: [email protected] (L. Shen). https://doi.org/10.1016/j.postharvbio.2018.11.012 Received 13 July 2018; Received in revised form 13 November 2018; Accepted 18 November 2018 0925-5214/ © 2018 Elsevier B.V. All rights reserved.
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overexpression of SlERF2 (Solanum lycopersicum ethylene response factor 2) has been reported to improve cold tolerance in tomato plant, and the antisense lines exhibited cold sensitive phenotype (Zhang and Huang, 2010). All these reports revealed that ethylene was positively correlated with chilling tolerance in tomato. Further studies are needed for insight into more specific relationship between ethylene biosynthesis and chilling tolerance in tomato fruit. Generally, ethylene biosynthesis occurs via methionine metabolism, in which the rate-limiting step is the conversion of S-adenosyl-L-methionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) via ACC synthase (ACS) (Adams and Yang, 1979). It has been previously reported that an ACS octuple mutant with extremely low levels of ethylene production was hypersensitive to freezing, accompanied by significantly lower AtCBF1/2 expression levels in Arabidopsis thaliana (Catala et al., 2014). In tomato, there is evidence that both transcript level and activity of ACS could be modulated under cold stress (Zhao et al., 2013). Although ethylene has been implicated in cold stress response, the exact role of ethylene biosynthesis in cold tolerance remained unclear in tomato. In this study, we used antisense SlACS2 and WT tomato fruit to investigate the role of ethylene biosynthesis in cold tolerance. Membrane damage, osmolytes content, antioxidant enzymes activities and relative expression of SlCBF1 were examined. Our results revealed that inhibiting ethylene biosynthesis led to decreased chilling tolerance and downregulated SlCBF1 expression, suggesting that ethylene biosynthesis plays a positive role in regulating chilling tolerance and SlCBF1 expression in tomato fruit. 2. Materials and methods 2.1. Fruit materials and treatments Tomato fruit (Solanum lycopersicum cv. Lichun) of WT and antisense SlACS2 (Luo et al., 1995) were harvested at mature green stage from a special vegetable base in Beijing, China, transported to laboratory immediately. All fruit without calyxes were selected for no blemishes or disease, with uniformity of shape, color (green), and size. Fruit were surface-disinfected with 2% (v/v) sodium hypochlorite for 2 min, washed with distilled water and air-dried. After that, all WT and antisense SlACS2 tomato fruit were stored at 4 °C up to 35 d, or 20 °C up to 24 h at ambient storage with 80–90 % relativity humidity (RH) in the dark. Six fruit from each group at random were sampled at 0, 2, 4, 8, 16 and 24 h during cold storage (4 °C) for ACS activity and SlCBF1 gene assay. Six fruit from each group at random were sampled on 0, 5, 15, 25, 35 d during cold storage (4 °C) for MDA content, proline content, soluble protein content and antioxidant enzymes analysis. Mesocarp from fruit equator area was cut into small pieces, frozen in liquid nitrogen rapidly and stored at −80 °C until analysis. Eight fruit chosen from each group were removed from cold on different days and hold at 20 °C for 3 d to develop CI symptoms (Ding et al., 2016). For ethylene production detecting, five fruit from each group were taken both at different hours and days. Three biological replicates were carried out in this experiment. 2.2. Determination of CI index CI was manifested by the percentage of affected area on fruit surface according to the method described by (Ding et al., 2016), all results were replicated three times. CI index was assessed according to the following four-stage scale: 0 = no pitting; 1 = pitting covering < 25% of the fruit surface; 2 = pitting covering < 50%, but > 25% of surface; 3 = pitting covering < 75%, but > 50% of surface and 4 = pitting covering > 75% of surface (Figure S1). The average extent of CI index was calculated using the following formula: CI index (%) = {∑[(CI level) × (Number of fruit at the CI level)]/(Total number of fruit) × 4} × 100%.
(caption on next page)
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Fig. 1. Effects of cold stress on (A) endogenous ethylene biosynthesis, (B) ACC content, (C) ACS activity, and (D) SlCBF1 expression in mature green WT tomato fruit. Blue shadow represents the time when the first peak of SlCBF1 expression and ACC content, ACS activity increased sharply at 2 h with cold stress. Pink shadow represents the time when the second peak of SlCBF1 expression and ethylene production, ACS activity peaked at 8 h with cold stress. Data represent means ± S.D., n = 3. Values followed by different letters represent significant differences at P < 0.05 level (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
replicated three times. 2.5. Determination of MDA content and ion leakage Content of malondialdehyde (MDA) was measured by thiobarbituric acid using the method of Ding et al. (2007). The amount of MDA was calculated from the extinction coefficient of 0.0155 μmol L−1. MDA content was calculated based on fresh weight, and expressed as μmol kg−1. Ion leakage was measured immediately from the mesocarp tissue before chilling (time 0) and on day 5, 15, 25, 35 after chilling treatment, using the method of Zhao et al. (2009b). All results were replicated three times.
2.3. Determination of ethylene production Ethylene was determined using the method described by McKeon et al. (1982), with some modifications. Ethylene production rate was determined by incubating five fruit from each treatment in a 9 L air tight chamber for 30 min under atmospheric pressure. A 1 mL of gas sample from headspace was withdrawn using a gas-tight syringe and injected into a gas chromatograph (GC- 4000 A, East& West Analytical, Beijing, China) equipped with a GDX-502 column and a flame ionization detector. The column temperature was 60 °C and the injection temperature was 120 °C. The flow rates used for nitrogen carrier gas, air and hydrogen were 0.83 mL s−1, 6.67 mL s−1 and 0.75 mL s−1. Ethylene production was calculated based on fresh weight, and expressed as pmol kg−1 s−1. All results were replicated three times.
2.6. Determination of proline and soluble protein content Proline measurement was carried out using the acid ninhydrin method described by Bates et al. (1973), with some modifications. Proline Content was calculated based on fresh weight, and expressed as ng kg−1. Soluble protein content was determined by absorbance at 595 nm using the method of Bradford (1976), with bovine serum albumin as a standard. The protein content was calculated based on fresh weight, and expressed as μg kg−1. All results were replicated three times. 2.7. Determination of antioxidant enzymes The activities of antioxidant enzymes in tomato fruit were calculated based on soluble protein content, and expressed as U kg−1. All results were replicated three times. Frozen pericarp tissue (0.5 g) was homogenized in 5 mL of 100 mM sodium phosphate buffer (pH 7.0, containing 5% (w/v) polyvinyl pyrrolidone, and 1 mM EDTA) (Zhao et al., 2011). The homogenates were centrifuged at 12,000 ×g for 10 min at 4 °C. The aliquots of supernatant were collected for antioxidant enzyme assays. Superoxide dismutase (SOD, EC: 1.15.1.1)
2.4. Determination of ACC content and ACS activity ACC content was determined using the method described by Lizada and Yang (1979), which was expressed as μmol kg−1. Extract for ACS activity determination was prepared according to the method described by Boller et al. (1979), and determined by the method of Lizada and Yang (1979), which was expressed as nmol kg−1 s−1. Both ACC content and ACS activity were calculated based on fresh weight. All results were
Fig. 2. Effects of antisense SlACS2 on (A) ethylene biosynthesis, (B) chilling injury index, chilling phenotype after (C) 5 d and (D) 25d exposure to cold stress in mature green tomato fruit. The fruit were obtained after storage at 4 °C for 5, 15, 25 and 35 d plus 3 d at 20 °C. Data represent means ± S.D., n = 3. Values followed by different letters represent significant differences at P < 0.05 level (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 141
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Fig. 3. Effects of antisense SlACS2 on (A) MDA content, (B) ion leakage, (C) proline content, and (D) soluble protein content in mature green tomato fruit under cold stress. The fruit were sampled before chilling treatment (time 0) and on 5, 15, 25, and 35 d at 4 °C. Data represent means ± S.D., n = 3. Values followed by different letters represent significant differences at P < 0.05 level (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
2.9. Statistical analysis
activity was analyzed according to Giannopolitis and Ries (1977), by measuring the ability to inhibit the photochemical reduction of nitroblue tetrazolium at 560 nm. Catalase (CAT, EC 1.11.1.6) activity was assayed using the method described by Larrigaudière et al. (2004), by recording the consumption of hydrogen peroxide at 240 nm. Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined by the method of Nakano and Asada (1981), by monitoring the decrease in absorbance of ascorbic acid at 290 nm.
Data were expressed as means ± SD. Statistical analyses were conducted with the use of statistical analysis software SPSS 20.0 (IBM Corp., Armonk, NY). Data were also analysed for the effects of both antisense SlACS2 and time using a two-way analysis of variance (ANOVA) followed by paired t-test, P-value < 0.05 was considered significant. Pearson’s correlation analysis was performed to determine the correlations between the ethylene biosynthesis and chilling-related indexes.
2.8. Quantitative real-time PCR (qRT-PCR) analysis
3. Results
Total RNA in 0.15 g of frozen pericarp tissue was extracted using an EasyPure Plant RNA Kit (Beijing Transgen Biotech Co. Ltd., Beijing, China). Total RNA was dissolved in 30 μL of RNase-free water, and was quantified with a spectrophotometer (NanoDrop Technologies, Inc.). First-strand cDNA was synthesized from 2 μg of RNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (Beijing Transgen Biotech Co. Ltd., Beijing, China) and stored at -20 °C. Real-time PCR was carried out in a Bio-Rad CFX96 real-time PCR system (Bio-Rad, USA), with a TransStart Top Green qPCR SuperMix (Beijing Transgen Biotech Co. Ltd., Beijing, China). 1 μL of cDNA from each sample was used in 10 μL volume per reaction and all assays were run in triplicate. The qPCR amplification protocol parameters were as follows: initial denaturation at 94 °C for 30 s, followed by 40 cycles of 5 s at 94 °C, 15 s at 60 °C, and 15 s at 72 °C. The threshold cycle (Ct) value was confirmed by melting-curve analysis, the relative expression of specific gene was normalised to the SlUbi3 Ct value and calculated using the 2−△△Ct method. The primer sequences used for qRT-PCR analysis were listed in Table S1.
3.1. Effects of cold stress on ethylene biosynthesis and SlCBF1 expression in tomato fruit To determine whether ethylene biosynthesis in WT tomato fruit could be induced by cold stress, ethylene production, ACC content and ACS activity were analyzed both at room temperature (RT) and at low temperature (4 °C). After chilling treatment, WT tomato fruit showed a remarkably higher ethylene production at 4–8 h compared with the RT group, which reached a maximum of 1.53 pmol kg−1 s−1 at 8 h (Fig. 1A, P < 0.05). ACC content at cold storage was significantly higher than RT at 2–8 h, which peaked at 2 h with a maximum of 0.27μmol kg−1 (Fig. 1B, P < 0.05). ACS activity exhibited fluctuating changes under cold stress, and two peaks of ACS activity were detected at 2 and 8 h after chilling treatment, which showed large differences from RT (Fig. 1C, P < 0.05). Moreover, there are no significant trend changes in SlCBF1 expression during RT storage, while SlCBF1 142
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3.2. Effects of ethylene biosynthesis on chilling injury in cold stored tomato fruit Antisense SlACS2 and WT tomato fruit were used to determine whether inhibiting ethylene biosynthesis could affect cold tolerance. Results showed that significant differences in ethylene levels were found between cold stored WT and antisense SlACS2 fruit. Ethylene content in WT fruit reached a maximum of 0.59 pmol kg−1 s−1 after 15 d cold storage and then gradually declined. In contrast, antisense SlACS2 fruit showed a decrease in ethylene production from day 5, and ethylene levels were below 0.17 pmol kg−1 s−1 in the whole storage (Fig. 2A, P < 0.05). Chilling injury was observed on the surface of both WT and transgenic fruit, which increased with the storage time. WT fruit showed a reduction in CI index to 24.8%, 28.8% and 32.0% of that in antisense SlACS2 tomato fruit on day 5, 15 and 25, respectively (Fig. 2B, P < 0.05). Color appearance in WT tomato fruit showed incipient red or yellow coloration after 5 d cold storage plus 3 d at 20 °C, while little color change was found in antisense SlACS2 fruit (Fig. 2C). The percentage of fruit ripened normally was 50.0% in WT and 12.5% in antisense SlACS2 fruit on day 5, corresponding with the increased CI index (Fig. 2B and C, P < 0.05). Both WT and antisense SlACS2 tomato fruit developed decay spot on day 25, while the decay degree in WT fruit was less than that in antisense SlACS2 tomato fruit (Fig. 2D). These results suggested that inhibiting ethylene biosynthesis reduced tomato fruit chilling tolerance, which played a positive role in cold response. 3.3. Effects of ethylene biosynthesis on MDA content, ion leakage, proline content, and soluble protein content in cold stored tomato fruit The levels of MDA and ion leakage were elevated in both WT and antisense SlACS2 tomato fruit in the process of cold storage (Fig. 3A and B, P < 0.05). However, antisense SlACS2 fruit had higher MDA level than WT under cold condition. MDA content in transgenic fruit on day 5 accumulated to a maximum of 66.19 μmol kg−1, significantly higher than the peak value in WT (Fig. 3A, P < 0.05). Ion leakage in WT fruit was higher than that in transgenic fruit from the beginning, but after 5 d under chilling treatment, ion leakage in transgenic fruit increased rapidly and became higher than that in WT (Fig. 3B, P < 0.05). Proline content in two groups increased after exposure to chilling treatment (Fig. 3C, P < 0.05). Proline content in transgenic fruit increased sharply on day 5 and then declined. However, in WT fruit, proline content displayed a near-liner increase and peaked on 25 d at 34.26 ng kg−1 then declined, which was higher than that in transgenic fruit (Fig. 3C, P < 0.05). Before chilling, a significant difference in soluble protein content could be detected between two groups, and transgenic fruit exhibited significantly higher level than WT. During chilling treatment, the biggest difference in soluble protein content between two groups was shown on day 15, and the value in WT was 32.13% higher than that in transgenic fruit (Fig. 3D, P < 0.05)
Fig. 4. Effects of antisense SlACS2 on the activities of antioxidant enzymes: (A) APX, (B) CAT, and (C) SOD in mature green tomato fruit under cold stress. The fruit were sampled before chilling treatment (time 0) and on 5, 15, 25, and 35 d at 4 °C. Data represent means ± S.D., n = 3. Values followed by different letters represent significant differences at P < 0.05 level (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3.4. Effects of ethylene biosynthesis on APX, CAT, and SOD activities in cold stored tomato fruit Antioxidant enzymes activities including APX, CAT, and SOD were significantly lower in antisense SlACS2 tomato fruit than those in WT (Fig. 4, P < 0.05). APX activity increased immediately after fruit exposed to chilling, and reached a peak on day 5, but the peak value in transgenic fruit was 17.3% lower than that in WT. During the whole chilling treatment, WT fruit showed a significant higher APX activity than that in transgenic fruit except the 25th day (Fig. 4A, P < 0.05). Changes in CAT activity were different between WT and transgenic fruit after chilling treatment, and CAT activity in transgenic fruit was significantly lower than that in WT (Fig. 4B, P < 0.05). SOD in WT fruit increased to a maximum of 11.19 U kg−1 on day 5, then declined to 7.66 U kg−1 on day 15 and kept the activity level with some fluctuation
expression was remarkably triggered under cold storage, which reached to an early peak at 2 h and a later peak at 8 h after chilling treatment (Fig. 1D, P < 0.05), coincident with the double-peak of ACS activity, and the first peak was coincident with the peak of ACC content, the second peak was coincident with the ethylene production too.
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Table 1 Pearson’s correlations among CI, ethylene content, MDA content, ion leakage, proline content, soluble protein content and antioxidant enzyme activities.
Chilling Index Ethylene Content MDA Content Ion Leakage Level Proline Content Soluble Protein Content APX Activity CAT Activity SOD Activity
Chilling Index
Ethylene Content
MDA Content
Ion Leakage Level
Proline Content
Soluble Protein Content
APX Activity
CAT Activity
SOD Activity
1.000
−0.531 1.000
0.686 −0.777* 1.000
0.900** −0.712* 0.79* 1.000
−0.926** 0.437 −0.415 −0.773* 1.000
0.231 0.292 −0.003 0.206 −0.164 1.000
−0.383 0.191 −0.542 −0.553 0.175 −0.312
−0.431 0.729* −0.730* −0.665 0.217 −0.285
0.262 0.209 −0.214 0.065 −0.465 −0.214
1.000
0.244 1.000
0.073 0.541 1.000
* Correlation is significant at the 0.05 level (2-tailed). ** Correlation is significant at the 0.01 level (2-tailed).
on day 25 and 35. SOD activity in transgenic fruit decreased sharply to the value of 4.7 U kg−1 on day 5, then reached a maximum of 9.20 U kg−1 on day 15. On day 25, SOD activity decreased again, and was 23.5% lower than that in WT (Fig. 4C, P < 0.05).
0.05). Generally, the change pattern of SlCBF1 expression was similar with the pattern of ethylene biosynthesis between two groups, and inhibiting ethylene biosynthesis downregulated SlCBF1 relative expression.
3.5. Correlation analysis of ethylene production and chilling-related indexes
3.7. Correlation analysis of ethylene biosynthesis and SlCBF1 expression
As seen in Table 1, positive correlations were found among CI-ion leakage, ethylene-CAT, MDA-ion leakage, and negative correlations were found among CI-proline, ion leakage-proline, MDA-CAT, ethyleneMDA and ethylene-ion leakage (P < 0.05). The close relationship between CI and ion leakage (r=0.9) could possibly be attributed to the fact that cell membrane was the first target of cold stress, and ion leakage could adversely affect the membrane integrity. Furthermore, the significant correlations among ethylene-MDA and ethylene-ion leakage might imply that endogenous ethylene contributed to alleviate chilling injury under cold condition, which might be due to that the activities of CAT could be activated by endogenous ethylene. These findings suggested a connection between ethylene production and chilling injury.
The correlation coefficients for ethylene content, ACC content, ACS activity and SlCBF1 expression were shown in Table 2. Significantly positive correlations were observed among ethylene-ACS, and ACCSlCBF1 under cold condition (P < 0.05).The significant correlation between ethylene content and ACS activity indicated that ACS activity played important role in endogenous ethylene production under cold condition (Table 2). Furthermore, the significant correlation between ACC content and SlCBF1 relative expression suggested that there might be a connection between ethylene biosynthesis and SlCBF1 relative expression. 4. Discussion Ethylene has long been reported to regulate cold tolerance in postharvest fruit, such as mango, grapefruit, kiwifruit, and tomato (Nair et al., 2004; Zhao et al., 2009a; 2009b, Lado et al., 2015; Joon, 2017). Although the functions of ethylene in cold response have been investigated, most studies focused on cold tolerance induced by exogenous ethylene treatment, the specific role of ethylene biosynthesis in tomato fruit cold response is still unclear, and it has never been reported using genetic method. In this study, we explored the role of ethylene biosynthesis in cold response by 1) investigating the effect of impaired ethylene biosynthesis on chilling tolerance, and 2) studying the correlation between SlCBF1 gene expression and ethylene biosynthesis after exposure to chilling. Firstly, to investigate the involvement of ethylene biosynthesis in tomato fruit chilling tolerance, antisense SlACS2 and WT tomato fruit were used, and CI symptoms in two groups were examined. Antisense SlACS2 fruit showed a lower ethylene production level than that in WT (Fig. 2A, P < 0.05). Ethylene production was inhibited in antisense SlACS2 tomato fruit as described by Oeller et al. (1991), indicating that ethylene biosynthesis was inhibited in antisense SlACS2 fruit under cold condition. Meanwhile, cold stress caused severer chilling phenotype in transgenic fruit than in WT, accompanied by higher CI index (Fig. 2B, P < 0.05), suggesting that blocking ethylene biosynthesis greatly decreased cold tolerance of tomato fruit during cold storage. Cell membrane damage is found commonly when plants are subjected to low temperatures. MDA, a degradation product of lipid peroxidation, is an important indicator of ROS (reactive oxygen species) destructive effects and membrane damage (Pallavi et al., 2012). Ion leakage level could also be used to evaluate the membrane integrity. Our results showed that MDA content and ion leakage level in
3.6. Effects of ethylene biosynthesis on SlCBF1 gene expression in cold stored tomato fruit Ethylene production between two groups during cold storage was quite different. Ethylene content in WT fruit increased rapidly from 4 h and reached a peak value of 1.53 pmol kg−1 s−1 at 8 h after chilling treatment, and increased again from 16 h to the end of the storage. However, ethylene production in antisense SlACS2 fruit gradually increased from 4 h to 16 h to reach the maximum, but it was 8 h later and the value was 33.0% lower than the peak in WT (Fig. 5A, P < 0.05). The changing pattern of ACC content in antisense SlACS2 fruit was similar to the WT under cold stress, which both peaked at 2 h, but ACC content in antisense SlACS2 fruit was lower compared with WT (Fig. 5B, P < 0.05). Changes in ACS activity were different between WT and antisense SlACS2 fruit. ACS activity in WT fruit exhibited an early peak at 2 h and a later peak at 8 h. In contrast, ACS activity in transgenic fruit showed only one peak at 16 h, which maintained at a lower level, with fluctuating changes along with storage time (Fig. 5C, P < 0.05). Though the increase in SlCBF1 gene expression was detected in both WT and antisense SlACS2 fruit after 2 h chilling treatment, gene expression level in WT fruit was 37.7% higher than that in transgenic fruit (Fig. 5D, P < 0.05). SlCBF1 expression in WT fruit increased from 4 h and peaked at 8 h, extremely significant higher than that in transgenic fruit and coincident with the time of both ethylene production peak and ACS activity peak appeared in WT fruit (Fig. 5, P < 0.05). Transgenic fruit showed relative lower SlCBF1 expression level and peaked at 2 and 16 h, the second peak of SlCBF1 expression was coincident with peaks of both endogenous ethylene production and ACS activity (Fig. 5, P < 144
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Fig. 5. Effects of antisense SlACS2 on (A) endogenous ethylene biosynthesis, (B) ACC content, (C) ACS activity, and (D) SlCBF1 expression in mature green tomato fruit under cold stress. Orange shadow represents the time when the first peak of SlCBF1 expression and ACC content, ACS activity peaked at 2 h in both WT and antisense SlACS2 tomato fruit. Pink shadow represents the time when the second peak of SlCBF1 expression and ethylene production, ACS activity peaked at 8 h in WT tomato fruit. Green shadow represents the time when the second peak of SlCBF1 expression and ethylene production, ACS activity peaked at 16 h in antisense SlACS2 tomato fruit. Data represent means ± S.D., n = 3. Values followed by different letters represent significant differences at P < 0.05 level (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Table 2 Pearson’s correlations among ethylene content, ACC content, ACS activitiy and SlCBF1 expression.
Ethylene Content ACC Content ACS Activity SlCBF1 Expression
Ethylene Content
ACC Content
1.000
0.752** 0.120 1.000
0.015 1.000
ACS Activity
SlCBF1 Expression 0.045 0.710** 0.452 1.000
** Correlation is significant at the 0.01 level (2-tailed).
proline helps in resisting chilling injury by maintaining the functional integrity of cellular membranes and directly participating in scavenging ROS in response to cold stress (Ben Rejeb et al., 2014; Ershadi et al., 2016). In this study, both proline and soluble protein levels were significantly lower in transgenic fruit under cold stress (Fig. 3C and D, P < 0.05), which were supported by previous study that tomato fruit with lower cold tolerance had lower levels of proline under cold stress (Zhao et al., 2009a). CI symptoms in tomato fruit such as surface lesions, discoloration, plant death and accelerated senescence, were often thought to be an outcome of ROS accumulation (Subramanian et al., 2016). To scavenge excess ROS, plants have evolved specific antioxidant enzymes such as APX, CAT, and SOD to protect themselves from oxidative damage, which play vital roles in regulating chilling resistance (Egea et al., 2010). In our study, APX, CAT, and SOD activities showed lower levels in antisense SlACS2 tomato fruit than those in WT (Fig. 4, P < 0.05). Qingrui Yang, (2011) also found that 1-MCP treatment significantly reduced endogenous ethylene production and chilling tolerance in sweet cherry, accompanied by suppression of antioxidant enzymes activities, which was consistent with our present study. Our results indicated that inhibiting ethylene biosynthesis could reduce antioxidant capacity, resulting in decreased chilling tolerance. SlCBF1 had a critical role in cold response, and SlCBF1 gene expression level was documented positively correlated with cold tolerance, and negatively correlated with CI symptoms in tomato fruit (Zhao et al., 2009a). In our current study, the relative expression of SlCBF1 was upregulated by chilling treatment and peaked at 2 h and 8 h in WT fruit (Fig. 1D, P < 0.05). There was a rapid elevation in ethylene production at 8 h, and a increase in ACC content at 2 h (Fig. 1A and B, P < 0.05), coincident with the second or the first peak of SlCBF1 expression (Fig. 1A, B and D, P < 0.05), which was consistent with previous study (Zhao et al., 2009b). Moreover, the double-peak expression mode of SlCBF1 gene was same as the double-peak of ACS activity, which exhibited two peaks at 2 h and 8 h after chilling treatment (Fig. 1C, P < 0.05). This coincidence suggested that there might be a correlation between SlCBF1 gene expression and endogenous ethylene biosynthesis. To confirm the correlation, SlCBF1 relative expression was compared between WT and antisense SlACS2 fruit. In our study, silence of SlACS2 significantly decreased ethylene production compared to WT fruit (Fig. 5A, P < 0.05). Meanwhile, the increased expression of
transgenic fruit were higher than those in WT under cold stress (Fig. 3A and B, P < 0.05), which suggested that inhibiting ethylene biosynthesis augmented membrane damage caused by chilling. Increased 145
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SlCBF1 was lower in transgenic fruit than in WT (Fig. 5D, P < 0.05). Moreover, ethylene production, ACS activity and SlCBF1 expression were all peaked at 16 h in antisense SlACS2 fruit, the same results were also observed in WT fruit, which all peaked at 8 h (Fig. 5, P < 0.05), and there was significant correlation between ACC content and SlCBF1 relative expression (Table 2, P < 0.01). These results revealed that endogenous ethylene biosynthesis could contribute to upregulate SlCBF1 expression, suggesting that ethylene biosynthesis was an important factor directly affecting SlCBF1 relative expression under cold condition. Similar conclusion has been drawn in Arabidopsis thaliana using genetic method, which implied that both endogenous ethylene biosynthesis and ethylene signaling transduction could regulate AtCBF genes expression under cold condition (Catala et al., 2014; Shi et al., 2012).
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5. Conclusion In conclusion, we find that ethylene biosynthesis is involved in regulating chilling tolerance and SlCBF1 relative expression in tomato fruit. Inhibiting ethylene biosynthesis in antisense SlACS2 fruit enhanced chilling injury. Moreover, the higher elevation in MDA content and ion leakage in antisense SlACS2 fruit, indicating that ethylene biosynthesis might play an important role in protecting cell membrane from oxidative damage caused by chilling treatment. In addition, activities of APX, CAT, and SOD were lower in antisense SlACS2 fruit, implying that antioxidant enzyme system was involved in chilling response induced by endogenous ethylene. These results suggest that the reduced chilling tolerance in antisense SlACS2 fruit could be correlated with the decrease in antioxidant capacity, which led to severer oxidative damage under chilling condition. Meanwhile, inhibited ethylene biosynthesis was partially correlated with the downregulation of SlCBF1 expression, which reveals that inhibiting ethylene biosynthesis could modulate SlCBF1 expression. Taken together, these results suggest that ethylene biosynthesis positively regulates cold tolerance and SlCBF1 relative expression in tomato fruit. Further investigations will focus on exploring the relationships between CBF signaling pathway and ethylene signaling transduction in tomato fruit under cold condition. Funding This work was supported by the National Natural Science Foundation of China (NO. 31371847, 31571893, and 31272215) Notes The authors declare no competing financial interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.postharvbio.2018.11. 012. References Adams, D.O., Yang, S.F., 1979. Ethylene biosynthesis - identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. U. S. A. 76, 170–174. Barry, C.S., Llop-Tous, M.I., Grierson, D., 2000. The regulation of 1-aminocyclopropane1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato. J. Plant Physiol. 123, 979–986. https://doi. org/10.1104/pp.123.3.979. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207. https://doi.org/10.1007/BF00018060. Ben Rejeb, K., Abdelly, C., Savouré, A., 2014. How reactive oxygen species and proline face stress together. Plant Physiol. Biochem. 80, 278–284. https://doi.org/10.1016/j. plaphy.2014.04.007.
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chilling resistance in tomato (Solanum lycopersicum Mill.) under low temperatures. PLoS One 11, e161592. https://doi.org/10.1371/journal.pone.0161592. Wang, L., Zhao, R., Zheng, Y., Chen, L., Li, R., Ma, J., Hong, X., Ma, P., Sheng, J., Shen, L., 2017. SlMAPK1/2/3 and antioxidant enzymes are associated with H2O2-induced chilling tolerance in tomato plants. J. Agric. Food Chem. 65, 6812–6820. https://doi. org/10.1021/acs.jafc. Xinhua Zhang, B.J.S.F., 2012. Methyl jasmonate alters arginine catabolism and improves postharvest chilling tolerance in cherry tomato. Postharvest Biol. Tec. 1, 160–167. https://doi.org/10.1016/j.postharvbio.2011.07.006. Zhang, Z., Huang, R., 2010. Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor TERF2/LeERF2 is modulated by ethylene biosynthesis. Plant Mol. Biol. 73, 241–249. https://doi.org/10.1007/s11103-0109609-4. Zhang, X., Fowler, S.G., Cheng, H.M., Lou, Y.G., Rhee, S.Y., Stockinger, E.J., Thomashow, M.F., 2004. Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis. Plant J. 39,
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Ethylene biosynthesis is involved in regulating chilling tolerance and SlCBF1 gene expression in tomato fruit Wenqing Yua, Jiping Shengb, Ruirui Zhaoa, Qin Wangc, Peihua Mac, Lin Shena,
T
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a
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China School of Agricultural Economics and Rural Development, Renmin University of China, Beijing, 100872, China c Department of Nutrition and Food Science, University of Maryland, College Park, MD20142, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Ethylene biosynthesis SlACS2 Cold tolerance SlCBF1 Tomato fruit
Ethylene plays positive role in cold response of tomato fruit. CBFs (C-repeat/dehydration-responsive element binding factors) have been proved important in regulating plant chilling tolerance. Till now, the role that ethylene biosynthesis played in chilling response of tomato fruit is still unclear. In this study, we used antisense SlACS2 and wild-type Lichun tomato fruit as inhibited- and normal- ethylene biosynthesis models, in order to investigate whether inhibiting ethylene biosynthesis could affect chilling tolerance and SlCBF1 relative expression in tomato fruit. Results showed that antisense SlACS2 tomato fruit suffered more chilling injury, accompanied by higher malondialdehyde content and ion leakage, less proline and soluble protein content, as well as lower antioxidant enzymes activities, suggesting that inhibiting ethylene biosynthesis reduced tomato fruit chilling tolerance. Furthermore, SlCBF1 expression in transgenic fruit was lower than in WT, the second peak of SlCBF1 expression was coincidence with the peaks of both endogenous ethylene production and ACS activity, and there was significant correlation between ACC content and SlCBF1 relative expression, which implied that endogenous ethylene biosynthesis could contribute to upregulate SlCBF1 expression under cold stress. These results revealed that ethylene biosynthesis played important roles in regulating tomato fruit chilling tolerance and SlCBF1 relative expression in tomato fruit.
1. Introduction Low-temperature storage is a widely used technique to prolong the life and preserve quality of fruit, while storage of tropical and subtropical fruit at temperatures below 12 °C raises the risk of chilling injury (CI) (Cruz-Mendívil et al., 2015). Tomato (Solanum lycopersicum) is originated from subtropical, which is a typical cold-sensitive fruit that has been widely used as a model to investigate cold-induced responses (Constán-Aguilar et al., 2014; Zhao et al., 2011). ICE–CBF−COR is currently the most investigated cold stress signaling pathway, in which CBFs (C-repeat/dehydration-responsive element binding factors) bind to cold-induced genes, played a pivotal role in the modulation of cold response (Stockinger et al., 1997). Three CBF genes have been identified in tomato, in which only the SlCBF1 gene could be effectively induced by cold stress (Zhang et al., 2004).
Previous studies revealed that SlCBF1 expression level was positively correlated with cold tolerance in tomato fruit (Zhao et al., 2009a). Apart from cold stress, several endogenous signal molecules could also regulate SlCBF1 expression, such as nitric oxide, hydrogen peroxide, methyl jasmonate, salicylic acid, gibberellin, and ethylene (Zhao et al., 2011; Ding et al., 2015; Wang et al., 2017; Xinhua Zhang, 2012; Ding et al., 2016; Zhao et al., 2009b), which contribute to increase tomato plant/fruit cold tolerance in a CBF-dependent manner. Ethylene biosynthesis mainly occurs in the process of fruit ripening or suffering stress, the latter is also called stress-induced ethylene, which has been thought to play an important role in stress response including cold stress response (Barry et al., 2000). Previous study has documented that blocking ethylene action with 1-MCP resulted in decreased cold tolerance, while treatment with ethephon led to increased cold tolerance in tomato fruit (Zhao et al., 2009b). Moreover,
Abbreviations: CBF, C-repeat/dehydration-responsive element binding factor; SlERF2, solanum lycopersicum ethylene response factor 2; SAM, S-adenosyl-L-methionine; ACC, 1-aminocyclopropane-1-carboxylic acid; ACS, ACC synthase; AVG, 2aminoethoxyvinyl glycine; CI, chilling injury; MDA, malondialdehyde; APX, ascorbate peroxidase; CAT, catalase; SOD, superoxide dismutases; Ct, threshold cycle; qRT-PCR, quantitative real-time PCR; WT, wild-type; RT, room temperature; ROS, reactive oxygen species ⁎ Corresponding author. E-mail address: [email protected] (L. Shen). https://doi.org/10.1016/j.postharvbio.2018.11.012 Received 13 July 2018; Received in revised form 13 November 2018; Accepted 18 November 2018 0925-5214/ © 2018 Elsevier B.V. All rights reserved.
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overexpression of SlERF2 (Solanum lycopersicum ethylene response factor 2) has been reported to improve cold tolerance in tomato plant, and the antisense lines exhibited cold sensitive phenotype (Zhang and Huang, 2010). All these reports revealed that ethylene was positively correlated with chilling tolerance in tomato. Further studies are needed for insight into more specific relationship between ethylene biosynthesis and chilling tolerance in tomato fruit. Generally, ethylene biosynthesis occurs via methionine metabolism, in which the rate-limiting step is the conversion of S-adenosyl-L-methionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC) via ACC synthase (ACS) (Adams and Yang, 1979). It has been previously reported that an ACS octuple mutant with extremely low levels of ethylene production was hypersensitive to freezing, accompanied by significantly lower AtCBF1/2 expression levels in Arabidopsis thaliana (Catala et al., 2014). In tomato, there is evidence that both transcript level and activity of ACS could be modulated under cold stress (Zhao et al., 2013). Although ethylene has been implicated in cold stress response, the exact role of ethylene biosynthesis in cold tolerance remained unclear in tomato. In this study, we used antisense SlACS2 and WT tomato fruit to investigate the role of ethylene biosynthesis in cold tolerance. Membrane damage, osmolytes content, antioxidant enzymes activities and relative expression of SlCBF1 were examined. Our results revealed that inhibiting ethylene biosynthesis led to decreased chilling tolerance and downregulated SlCBF1 expression, suggesting that ethylene biosynthesis plays a positive role in regulating chilling tolerance and SlCBF1 expression in tomato fruit. 2. Materials and methods 2.1. Fruit materials and treatments Tomato fruit (Solanum lycopersicum cv. Lichun) of WT and antisense SlACS2 (Luo et al., 1995) were harvested at mature green stage from a special vegetable base in Beijing, China, transported to laboratory immediately. All fruit without calyxes were selected for no blemishes or disease, with uniformity of shape, color (green), and size. Fruit were surface-disinfected with 2% (v/v) sodium hypochlorite for 2 min, washed with distilled water and air-dried. After that, all WT and antisense SlACS2 tomato fruit were stored at 4 °C up to 35 d, or 20 °C up to 24 h at ambient storage with 80–90 % relativity humidity (RH) in the dark. Six fruit from each group at random were sampled at 0, 2, 4, 8, 16 and 24 h during cold storage (4 °C) for ACS activity and SlCBF1 gene assay. Six fruit from each group at random were sampled on 0, 5, 15, 25, 35 d during cold storage (4 °C) for MDA content, proline content, soluble protein content and antioxidant enzymes analysis. Mesocarp from fruit equator area was cut into small pieces, frozen in liquid nitrogen rapidly and stored at −80 °C until analysis. Eight fruit chosen from each group were removed from cold on different days and hold at 20 °C for 3 d to develop CI symptoms (Ding et al., 2016). For ethylene production detecting, five fruit from each group were taken both at different hours and days. Three biological replicates were carried out in this experiment. 2.2. Determination of CI index CI was manifested by the percentage of affected area on fruit surface according to the method described by (Ding et al., 2016), all results were replicated three times. CI index was assessed according to the following four-stage scale: 0 = no pitting; 1 = pitting covering < 25% of the fruit surface; 2 = pitting covering < 50%, but > 25% of surface; 3 = pitting covering < 75%, but > 50% of surface and 4 = pitting covering > 75% of surface (Figure S1). The average extent of CI index was calculated using the following formula: CI index (%) = {∑[(CI level) × (Number of fruit at the CI level)]/(Total number of fruit) × 4} × 100%.
(caption on next page)
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Fig. 1. Effects of cold stress on (A) endogenous ethylene biosynthesis, (B) ACC content, (C) ACS activity, and (D) SlCBF1 expression in mature green WT tomato fruit. Blue shadow represents the time when the first peak of SlCBF1 expression and ACC content, ACS activity increased sharply at 2 h with cold stress. Pink shadow represents the time when the second peak of SlCBF1 expression and ethylene production, ACS activity peaked at 8 h with cold stress. Data represent means ± S.D., n = 3. Values followed by different letters represent significant differences at P < 0.05 level (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
replicated three times. 2.5. Determination of MDA content and ion leakage Content of malondialdehyde (MDA) was measured by thiobarbituric acid using the method of Ding et al. (2007). The amount of MDA was calculated from the extinction coefficient of 0.0155 μmol L−1. MDA content was calculated based on fresh weight, and expressed as μmol kg−1. Ion leakage was measured immediately from the mesocarp tissue before chilling (time 0) and on day 5, 15, 25, 35 after chilling treatment, using the method of Zhao et al. (2009b). All results were replicated three times.
2.3. Determination of ethylene production Ethylene was determined using the method described by McKeon et al. (1982), with some modifications. Ethylene production rate was determined by incubating five fruit from each treatment in a 9 L air tight chamber for 30 min under atmospheric pressure. A 1 mL of gas sample from headspace was withdrawn using a gas-tight syringe and injected into a gas chromatograph (GC- 4000 A, East& West Analytical, Beijing, China) equipped with a GDX-502 column and a flame ionization detector. The column temperature was 60 °C and the injection temperature was 120 °C. The flow rates used for nitrogen carrier gas, air and hydrogen were 0.83 mL s−1, 6.67 mL s−1 and 0.75 mL s−1. Ethylene production was calculated based on fresh weight, and expressed as pmol kg−1 s−1. All results were replicated three times.
2.6. Determination of proline and soluble protein content Proline measurement was carried out using the acid ninhydrin method described by Bates et al. (1973), with some modifications. Proline Content was calculated based on fresh weight, and expressed as ng kg−1. Soluble protein content was determined by absorbance at 595 nm using the method of Bradford (1976), with bovine serum albumin as a standard. The protein content was calculated based on fresh weight, and expressed as μg kg−1. All results were replicated three times. 2.7. Determination of antioxidant enzymes The activities of antioxidant enzymes in tomato fruit were calculated based on soluble protein content, and expressed as U kg−1. All results were replicated three times. Frozen pericarp tissue (0.5 g) was homogenized in 5 mL of 100 mM sodium phosphate buffer (pH 7.0, containing 5% (w/v) polyvinyl pyrrolidone, and 1 mM EDTA) (Zhao et al., 2011). The homogenates were centrifuged at 12,000 ×g for 10 min at 4 °C. The aliquots of supernatant were collected for antioxidant enzyme assays. Superoxide dismutase (SOD, EC: 1.15.1.1)
2.4. Determination of ACC content and ACS activity ACC content was determined using the method described by Lizada and Yang (1979), which was expressed as μmol kg−1. Extract for ACS activity determination was prepared according to the method described by Boller et al. (1979), and determined by the method of Lizada and Yang (1979), which was expressed as nmol kg−1 s−1. Both ACC content and ACS activity were calculated based on fresh weight. All results were
Fig. 2. Effects of antisense SlACS2 on (A) ethylene biosynthesis, (B) chilling injury index, chilling phenotype after (C) 5 d and (D) 25d exposure to cold stress in mature green tomato fruit. The fruit were obtained after storage at 4 °C for 5, 15, 25 and 35 d plus 3 d at 20 °C. Data represent means ± S.D., n = 3. Values followed by different letters represent significant differences at P < 0.05 level (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 141
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Fig. 3. Effects of antisense SlACS2 on (A) MDA content, (B) ion leakage, (C) proline content, and (D) soluble protein content in mature green tomato fruit under cold stress. The fruit were sampled before chilling treatment (time 0) and on 5, 15, 25, and 35 d at 4 °C. Data represent means ± S.D., n = 3. Values followed by different letters represent significant differences at P < 0.05 level (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
2.9. Statistical analysis
activity was analyzed according to Giannopolitis and Ries (1977), by measuring the ability to inhibit the photochemical reduction of nitroblue tetrazolium at 560 nm. Catalase (CAT, EC 1.11.1.6) activity was assayed using the method described by Larrigaudière et al. (2004), by recording the consumption of hydrogen peroxide at 240 nm. Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined by the method of Nakano and Asada (1981), by monitoring the decrease in absorbance of ascorbic acid at 290 nm.
Data were expressed as means ± SD. Statistical analyses were conducted with the use of statistical analysis software SPSS 20.0 (IBM Corp., Armonk, NY). Data were also analysed for the effects of both antisense SlACS2 and time using a two-way analysis of variance (ANOVA) followed by paired t-test, P-value < 0.05 was considered significant. Pearson’s correlation analysis was performed to determine the correlations between the ethylene biosynthesis and chilling-related indexes.
2.8. Quantitative real-time PCR (qRT-PCR) analysis
3. Results
Total RNA in 0.15 g of frozen pericarp tissue was extracted using an EasyPure Plant RNA Kit (Beijing Transgen Biotech Co. Ltd., Beijing, China). Total RNA was dissolved in 30 μL of RNase-free water, and was quantified with a spectrophotometer (NanoDrop Technologies, Inc.). First-strand cDNA was synthesized from 2 μg of RNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (Beijing Transgen Biotech Co. Ltd., Beijing, China) and stored at -20 °C. Real-time PCR was carried out in a Bio-Rad CFX96 real-time PCR system (Bio-Rad, USA), with a TransStart Top Green qPCR SuperMix (Beijing Transgen Biotech Co. Ltd., Beijing, China). 1 μL of cDNA from each sample was used in 10 μL volume per reaction and all assays were run in triplicate. The qPCR amplification protocol parameters were as follows: initial denaturation at 94 °C for 30 s, followed by 40 cycles of 5 s at 94 °C, 15 s at 60 °C, and 15 s at 72 °C. The threshold cycle (Ct) value was confirmed by melting-curve analysis, the relative expression of specific gene was normalised to the SlUbi3 Ct value and calculated using the 2−△△Ct method. The primer sequences used for qRT-PCR analysis were listed in Table S1.
3.1. Effects of cold stress on ethylene biosynthesis and SlCBF1 expression in tomato fruit To determine whether ethylene biosynthesis in WT tomato fruit could be induced by cold stress, ethylene production, ACC content and ACS activity were analyzed both at room temperature (RT) and at low temperature (4 °C). After chilling treatment, WT tomato fruit showed a remarkably higher ethylene production at 4–8 h compared with the RT group, which reached a maximum of 1.53 pmol kg−1 s−1 at 8 h (Fig. 1A, P < 0.05). ACC content at cold storage was significantly higher than RT at 2–8 h, which peaked at 2 h with a maximum of 0.27μmol kg−1 (Fig. 1B, P < 0.05). ACS activity exhibited fluctuating changes under cold stress, and two peaks of ACS activity were detected at 2 and 8 h after chilling treatment, which showed large differences from RT (Fig. 1C, P < 0.05). Moreover, there are no significant trend changes in SlCBF1 expression during RT storage, while SlCBF1 142
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3.2. Effects of ethylene biosynthesis on chilling injury in cold stored tomato fruit Antisense SlACS2 and WT tomato fruit were used to determine whether inhibiting ethylene biosynthesis could affect cold tolerance. Results showed that significant differences in ethylene levels were found between cold stored WT and antisense SlACS2 fruit. Ethylene content in WT fruit reached a maximum of 0.59 pmol kg−1 s−1 after 15 d cold storage and then gradually declined. In contrast, antisense SlACS2 fruit showed a decrease in ethylene production from day 5, and ethylene levels were below 0.17 pmol kg−1 s−1 in the whole storage (Fig. 2A, P < 0.05). Chilling injury was observed on the surface of both WT and transgenic fruit, which increased with the storage time. WT fruit showed a reduction in CI index to 24.8%, 28.8% and 32.0% of that in antisense SlACS2 tomato fruit on day 5, 15 and 25, respectively (Fig. 2B, P < 0.05). Color appearance in WT tomato fruit showed incipient red or yellow coloration after 5 d cold storage plus 3 d at 20 °C, while little color change was found in antisense SlACS2 fruit (Fig. 2C). The percentage of fruit ripened normally was 50.0% in WT and 12.5% in antisense SlACS2 fruit on day 5, corresponding with the increased CI index (Fig. 2B and C, P < 0.05). Both WT and antisense SlACS2 tomato fruit developed decay spot on day 25, while the decay degree in WT fruit was less than that in antisense SlACS2 tomato fruit (Fig. 2D). These results suggested that inhibiting ethylene biosynthesis reduced tomato fruit chilling tolerance, which played a positive role in cold response. 3.3. Effects of ethylene biosynthesis on MDA content, ion leakage, proline content, and soluble protein content in cold stored tomato fruit The levels of MDA and ion leakage were elevated in both WT and antisense SlACS2 tomato fruit in the process of cold storage (Fig. 3A and B, P < 0.05). However, antisense SlACS2 fruit had higher MDA level than WT under cold condition. MDA content in transgenic fruit on day 5 accumulated to a maximum of 66.19 μmol kg−1, significantly higher than the peak value in WT (Fig. 3A, P < 0.05). Ion leakage in WT fruit was higher than that in transgenic fruit from the beginning, but after 5 d under chilling treatment, ion leakage in transgenic fruit increased rapidly and became higher than that in WT (Fig. 3B, P < 0.05). Proline content in two groups increased after exposure to chilling treatment (Fig. 3C, P < 0.05). Proline content in transgenic fruit increased sharply on day 5 and then declined. However, in WT fruit, proline content displayed a near-liner increase and peaked on 25 d at 34.26 ng kg−1 then declined, which was higher than that in transgenic fruit (Fig. 3C, P < 0.05). Before chilling, a significant difference in soluble protein content could be detected between two groups, and transgenic fruit exhibited significantly higher level than WT. During chilling treatment, the biggest difference in soluble protein content between two groups was shown on day 15, and the value in WT was 32.13% higher than that in transgenic fruit (Fig. 3D, P < 0.05)
Fig. 4. Effects of antisense SlACS2 on the activities of antioxidant enzymes: (A) APX, (B) CAT, and (C) SOD in mature green tomato fruit under cold stress. The fruit were sampled before chilling treatment (time 0) and on 5, 15, 25, and 35 d at 4 °C. Data represent means ± S.D., n = 3. Values followed by different letters represent significant differences at P < 0.05 level (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3.4. Effects of ethylene biosynthesis on APX, CAT, and SOD activities in cold stored tomato fruit Antioxidant enzymes activities including APX, CAT, and SOD were significantly lower in antisense SlACS2 tomato fruit than those in WT (Fig. 4, P < 0.05). APX activity increased immediately after fruit exposed to chilling, and reached a peak on day 5, but the peak value in transgenic fruit was 17.3% lower than that in WT. During the whole chilling treatment, WT fruit showed a significant higher APX activity than that in transgenic fruit except the 25th day (Fig. 4A, P < 0.05). Changes in CAT activity were different between WT and transgenic fruit after chilling treatment, and CAT activity in transgenic fruit was significantly lower than that in WT (Fig. 4B, P < 0.05). SOD in WT fruit increased to a maximum of 11.19 U kg−1 on day 5, then declined to 7.66 U kg−1 on day 15 and kept the activity level with some fluctuation
expression was remarkably triggered under cold storage, which reached to an early peak at 2 h and a later peak at 8 h after chilling treatment (Fig. 1D, P < 0.05), coincident with the double-peak of ACS activity, and the first peak was coincident with the peak of ACC content, the second peak was coincident with the ethylene production too.
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Table 1 Pearson’s correlations among CI, ethylene content, MDA content, ion leakage, proline content, soluble protein content and antioxidant enzyme activities.
Chilling Index Ethylene Content MDA Content Ion Leakage Level Proline Content Soluble Protein Content APX Activity CAT Activity SOD Activity
Chilling Index
Ethylene Content
MDA Content
Ion Leakage Level
Proline Content
Soluble Protein Content
APX Activity
CAT Activity
SOD Activity
1.000
−0.531 1.000
0.686 −0.777* 1.000
0.900** −0.712* 0.79* 1.000
−0.926** 0.437 −0.415 −0.773* 1.000
0.231 0.292 −0.003 0.206 −0.164 1.000
−0.383 0.191 −0.542 −0.553 0.175 −0.312
−0.431 0.729* −0.730* −0.665 0.217 −0.285
0.262 0.209 −0.214 0.065 −0.465 −0.214
1.000
0.244 1.000
0.073 0.541 1.000
* Correlation is significant at the 0.05 level (2-tailed). ** Correlation is significant at the 0.01 level (2-tailed).
on day 25 and 35. SOD activity in transgenic fruit decreased sharply to the value of 4.7 U kg−1 on day 5, then reached a maximum of 9.20 U kg−1 on day 15. On day 25, SOD activity decreased again, and was 23.5% lower than that in WT (Fig. 4C, P < 0.05).
0.05). Generally, the change pattern of SlCBF1 expression was similar with the pattern of ethylene biosynthesis between two groups, and inhibiting ethylene biosynthesis downregulated SlCBF1 relative expression.
3.5. Correlation analysis of ethylene production and chilling-related indexes
3.7. Correlation analysis of ethylene biosynthesis and SlCBF1 expression
As seen in Table 1, positive correlations were found among CI-ion leakage, ethylene-CAT, MDA-ion leakage, and negative correlations were found among CI-proline, ion leakage-proline, MDA-CAT, ethyleneMDA and ethylene-ion leakage (P < 0.05). The close relationship between CI and ion leakage (r=0.9) could possibly be attributed to the fact that cell membrane was the first target of cold stress, and ion leakage could adversely affect the membrane integrity. Furthermore, the significant correlations among ethylene-MDA and ethylene-ion leakage might imply that endogenous ethylene contributed to alleviate chilling injury under cold condition, which might be due to that the activities of CAT could be activated by endogenous ethylene. These findings suggested a connection between ethylene production and chilling injury.
The correlation coefficients for ethylene content, ACC content, ACS activity and SlCBF1 expression were shown in Table 2. Significantly positive correlations were observed among ethylene-ACS, and ACCSlCBF1 under cold condition (P < 0.05).The significant correlation between ethylene content and ACS activity indicated that ACS activity played important role in endogenous ethylene production under cold condition (Table 2). Furthermore, the significant correlation between ACC content and SlCBF1 relative expression suggested that there might be a connection between ethylene biosynthesis and SlCBF1 relative expression. 4. Discussion Ethylene has long been reported to regulate cold tolerance in postharvest fruit, such as mango, grapefruit, kiwifruit, and tomato (Nair et al., 2004; Zhao et al., 2009a; 2009b, Lado et al., 2015; Joon, 2017). Although the functions of ethylene in cold response have been investigated, most studies focused on cold tolerance induced by exogenous ethylene treatment, the specific role of ethylene biosynthesis in tomato fruit cold response is still unclear, and it has never been reported using genetic method. In this study, we explored the role of ethylene biosynthesis in cold response by 1) investigating the effect of impaired ethylene biosynthesis on chilling tolerance, and 2) studying the correlation between SlCBF1 gene expression and ethylene biosynthesis after exposure to chilling. Firstly, to investigate the involvement of ethylene biosynthesis in tomato fruit chilling tolerance, antisense SlACS2 and WT tomato fruit were used, and CI symptoms in two groups were examined. Antisense SlACS2 fruit showed a lower ethylene production level than that in WT (Fig. 2A, P < 0.05). Ethylene production was inhibited in antisense SlACS2 tomato fruit as described by Oeller et al. (1991), indicating that ethylene biosynthesis was inhibited in antisense SlACS2 fruit under cold condition. Meanwhile, cold stress caused severer chilling phenotype in transgenic fruit than in WT, accompanied by higher CI index (Fig. 2B, P < 0.05), suggesting that blocking ethylene biosynthesis greatly decreased cold tolerance of tomato fruit during cold storage. Cell membrane damage is found commonly when plants are subjected to low temperatures. MDA, a degradation product of lipid peroxidation, is an important indicator of ROS (reactive oxygen species) destructive effects and membrane damage (Pallavi et al., 2012). Ion leakage level could also be used to evaluate the membrane integrity. Our results showed that MDA content and ion leakage level in
3.6. Effects of ethylene biosynthesis on SlCBF1 gene expression in cold stored tomato fruit Ethylene production between two groups during cold storage was quite different. Ethylene content in WT fruit increased rapidly from 4 h and reached a peak value of 1.53 pmol kg−1 s−1 at 8 h after chilling treatment, and increased again from 16 h to the end of the storage. However, ethylene production in antisense SlACS2 fruit gradually increased from 4 h to 16 h to reach the maximum, but it was 8 h later and the value was 33.0% lower than the peak in WT (Fig. 5A, P < 0.05). The changing pattern of ACC content in antisense SlACS2 fruit was similar to the WT under cold stress, which both peaked at 2 h, but ACC content in antisense SlACS2 fruit was lower compared with WT (Fig. 5B, P < 0.05). Changes in ACS activity were different between WT and antisense SlACS2 fruit. ACS activity in WT fruit exhibited an early peak at 2 h and a later peak at 8 h. In contrast, ACS activity in transgenic fruit showed only one peak at 16 h, which maintained at a lower level, with fluctuating changes along with storage time (Fig. 5C, P < 0.05). Though the increase in SlCBF1 gene expression was detected in both WT and antisense SlACS2 fruit after 2 h chilling treatment, gene expression level in WT fruit was 37.7% higher than that in transgenic fruit (Fig. 5D, P < 0.05). SlCBF1 expression in WT fruit increased from 4 h and peaked at 8 h, extremely significant higher than that in transgenic fruit and coincident with the time of both ethylene production peak and ACS activity peak appeared in WT fruit (Fig. 5, P < 0.05). Transgenic fruit showed relative lower SlCBF1 expression level and peaked at 2 and 16 h, the second peak of SlCBF1 expression was coincident with peaks of both endogenous ethylene production and ACS activity (Fig. 5, P < 144
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Fig. 5. Effects of antisense SlACS2 on (A) endogenous ethylene biosynthesis, (B) ACC content, (C) ACS activity, and (D) SlCBF1 expression in mature green tomato fruit under cold stress. Orange shadow represents the time when the first peak of SlCBF1 expression and ACC content, ACS activity peaked at 2 h in both WT and antisense SlACS2 tomato fruit. Pink shadow represents the time when the second peak of SlCBF1 expression and ethylene production, ACS activity peaked at 8 h in WT tomato fruit. Green shadow represents the time when the second peak of SlCBF1 expression and ethylene production, ACS activity peaked at 16 h in antisense SlACS2 tomato fruit. Data represent means ± S.D., n = 3. Values followed by different letters represent significant differences at P < 0.05 level (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Table 2 Pearson’s correlations among ethylene content, ACC content, ACS activitiy and SlCBF1 expression.
Ethylene Content ACC Content ACS Activity SlCBF1 Expression
Ethylene Content
ACC Content
1.000
0.752** 0.120 1.000
0.015 1.000
ACS Activity
SlCBF1 Expression 0.045 0.710** 0.452 1.000
** Correlation is significant at the 0.01 level (2-tailed).
proline helps in resisting chilling injury by maintaining the functional integrity of cellular membranes and directly participating in scavenging ROS in response to cold stress (Ben Rejeb et al., 2014; Ershadi et al., 2016). In this study, both proline and soluble protein levels were significantly lower in transgenic fruit under cold stress (Fig. 3C and D, P < 0.05), which were supported by previous study that tomato fruit with lower cold tolerance had lower levels of proline under cold stress (Zhao et al., 2009a). CI symptoms in tomato fruit such as surface lesions, discoloration, plant death and accelerated senescence, were often thought to be an outcome of ROS accumulation (Subramanian et al., 2016). To scavenge excess ROS, plants have evolved specific antioxidant enzymes such as APX, CAT, and SOD to protect themselves from oxidative damage, which play vital roles in regulating chilling resistance (Egea et al., 2010). In our study, APX, CAT, and SOD activities showed lower levels in antisense SlACS2 tomato fruit than those in WT (Fig. 4, P < 0.05). Qingrui Yang, (2011) also found that 1-MCP treatment significantly reduced endogenous ethylene production and chilling tolerance in sweet cherry, accompanied by suppression of antioxidant enzymes activities, which was consistent with our present study. Our results indicated that inhibiting ethylene biosynthesis could reduce antioxidant capacity, resulting in decreased chilling tolerance. SlCBF1 had a critical role in cold response, and SlCBF1 gene expression level was documented positively correlated with cold tolerance, and negatively correlated with CI symptoms in tomato fruit (Zhao et al., 2009a). In our current study, the relative expression of SlCBF1 was upregulated by chilling treatment and peaked at 2 h and 8 h in WT fruit (Fig. 1D, P < 0.05). There was a rapid elevation in ethylene production at 8 h, and a increase in ACC content at 2 h (Fig. 1A and B, P < 0.05), coincident with the second or the first peak of SlCBF1 expression (Fig. 1A, B and D, P < 0.05), which was consistent with previous study (Zhao et al., 2009b). Moreover, the double-peak expression mode of SlCBF1 gene was same as the double-peak of ACS activity, which exhibited two peaks at 2 h and 8 h after chilling treatment (Fig. 1C, P < 0.05). This coincidence suggested that there might be a correlation between SlCBF1 gene expression and endogenous ethylene biosynthesis. To confirm the correlation, SlCBF1 relative expression was compared between WT and antisense SlACS2 fruit. In our study, silence of SlACS2 significantly decreased ethylene production compared to WT fruit (Fig. 5A, P < 0.05). Meanwhile, the increased expression of
transgenic fruit were higher than those in WT under cold stress (Fig. 3A and B, P < 0.05), which suggested that inhibiting ethylene biosynthesis augmented membrane damage caused by chilling. Increased 145
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SlCBF1 was lower in transgenic fruit than in WT (Fig. 5D, P < 0.05). Moreover, ethylene production, ACS activity and SlCBF1 expression were all peaked at 16 h in antisense SlACS2 fruit, the same results were also observed in WT fruit, which all peaked at 8 h (Fig. 5, P < 0.05), and there was significant correlation between ACC content and SlCBF1 relative expression (Table 2, P < 0.01). These results revealed that endogenous ethylene biosynthesis could contribute to upregulate SlCBF1 expression, suggesting that ethylene biosynthesis was an important factor directly affecting SlCBF1 relative expression under cold condition. Similar conclusion has been drawn in Arabidopsis thaliana using genetic method, which implied that both endogenous ethylene biosynthesis and ethylene signaling transduction could regulate AtCBF genes expression under cold condition (Catala et al., 2014; Shi et al., 2012).
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5. Conclusion In conclusion, we find that ethylene biosynthesis is involved in regulating chilling tolerance and SlCBF1 relative expression in tomato fruit. Inhibiting ethylene biosynthesis in antisense SlACS2 fruit enhanced chilling injury. Moreover, the higher elevation in MDA content and ion leakage in antisense SlACS2 fruit, indicating that ethylene biosynthesis might play an important role in protecting cell membrane from oxidative damage caused by chilling treatment. In addition, activities of APX, CAT, and SOD were lower in antisense SlACS2 fruit, implying that antioxidant enzyme system was involved in chilling response induced by endogenous ethylene. These results suggest that the reduced chilling tolerance in antisense SlACS2 fruit could be correlated with the decrease in antioxidant capacity, which led to severer oxidative damage under chilling condition. Meanwhile, inhibited ethylene biosynthesis was partially correlated with the downregulation of SlCBF1 expression, which reveals that inhibiting ethylene biosynthesis could modulate SlCBF1 expression. Taken together, these results suggest that ethylene biosynthesis positively regulates cold tolerance and SlCBF1 relative expression in tomato fruit. Further investigations will focus on exploring the relationships between CBF signaling pathway and ethylene signaling transduction in tomato fruit under cold condition. Funding This work was supported by the National Natural Science Foundation of China (NO. 31371847, 31571893, and 31272215) Notes The authors declare no competing financial interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.postharvbio.2018.11. 012. References Adams, D.O., Yang, S.F., 1979. Ethylene biosynthesis - identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. U. S. A. 76, 170–174. Barry, C.S., Llop-Tous, M.I., Grierson, D., 2000. The regulation of 1-aminocyclopropane1-carboxylic acid synthase gene expression during the transition from system-1 to system-2 ethylene synthesis in tomato. J. Plant Physiol. 123, 979–986. https://doi. org/10.1104/pp.123.3.979. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207. https://doi.org/10.1007/BF00018060. Ben Rejeb, K., Abdelly, C., Savouré, A., 2014. How reactive oxygen species and proline face stress together. Plant Physiol. Biochem. 80, 278–284. https://doi.org/10.1016/j. plaphy.2014.04.007.
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