Hydrogen Sulfide Inhibits Fruit Softening by Regulating Ethylene Synthesis and Signaling Pathway in Tomato (Solanum lycopersicum)

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  • 1 School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, China
  • | 2 Xuzhou Institute of Agricultural Sciences of the Xuhuai District of Jiangsu Province, Xuzhou 221131, China
  • | 3 School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, China
  • | 4 School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, China; Anhui Province Key Laboratory of Functional Compound Seasoning, Anhui Qiangwang Seasoning Food Co., Ltd., Jieshou 236500, China
  • | 5 School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, China

Hydrogen sulfide (H2S) has been proven to be a multifunctional signaling molecule in plants. In this study, we attempted to explore the effects of H2S on the climacteric fruit tomato during postharvest storage. H2S fumigation for 1 d was found to delay the peel color transition from green to red and decreased fruit firmness induced by ethylene. Further investigation showed that H2S fumigation downregulated the activities and gene expressions of cell wall–degrading enzymes pectin lyase (PL), polygalacturonase (PG), and cellulase. Furthermore, H2S fumigation downregulated the expression of ethylene biosynthesis genes SlACS2 and SlACS3. Ethylene treatment for 1 d was found to induce the expression of SlACO1, SlACO3, and SlACO4 genes, whereas the increase was significantly inhibited by H2S combined with ethylene. Furthermore, H2S decreased the transcript accumulation of ethylene receptor genes SlETR5 and SlETR6 and ethylene transcription factors SlCRF2 and SlERF2. The correlation analysis suggested that the fruit firmness was negatively correlated with ethylene biosynthesis and signaling pathway. The current study showed that exogenous H2S could inhibit the synthesis of endogenous ethylene and regulate ethylene signal transduction, thereby delaying fruit softening and the ripening process of tomato fruit during postharvest storage.

Abstract

Hydrogen sulfide (H2S) has been proven to be a multifunctional signaling molecule in plants. In this study, we attempted to explore the effects of H2S on the climacteric fruit tomato during postharvest storage. H2S fumigation for 1 d was found to delay the peel color transition from green to red and decreased fruit firmness induced by ethylene. Further investigation showed that H2S fumigation downregulated the activities and gene expressions of cell wall–degrading enzymes pectin lyase (PL), polygalacturonase (PG), and cellulase. Furthermore, H2S fumigation downregulated the expression of ethylene biosynthesis genes SlACS2 and SlACS3. Ethylene treatment for 1 d was found to induce the expression of SlACO1, SlACO3, and SlACO4 genes, whereas the increase was significantly inhibited by H2S combined with ethylene. Furthermore, H2S decreased the transcript accumulation of ethylene receptor genes SlETR5 and SlETR6 and ethylene transcription factors SlCRF2 and SlERF2. The correlation analysis suggested that the fruit firmness was negatively correlated with ethylene biosynthesis and signaling pathway. The current study showed that exogenous H2S could inhibit the synthesis of endogenous ethylene and regulate ethylene signal transduction, thereby delaying fruit softening and the ripening process of tomato fruit during postharvest storage.

Tomato (Solanum lycopersicum L.) is an important fruit for human consumption because it provides nutrients like flavor compounds, vitamins, and fiber (Beecher, 1998). Postharvest ripening and senescence involve changes in metabolism and gene expression, which further affect sensory attributes, flesh texture, fruit nutritional quality, and market acceptance. Fruit ripening and senescence are accompanied by texture softening, which is one of the most important factors regarding fruit quality and consumer acceptability (Giovannoni, 2007; Grierson et al., 1986; Musse et al., 2009). Fruit softening is a complex process that results from loosening of the cell wall. Many enzymes such as pectinmethylesterase, polygalacturonase, cellulase, and pectin lyase catalyze the modification and degradation of cell wall components, including pectin, hemicellulose, and others (Fischer and Bennett, 1991; Gwanpua et al., 2016; Thompson et al., 1998). Therefore, delaying fruit softening will maintain the texture of fruit and improve the market values.

Ethylene is an important phytohormone in plants and is involved in many aspects of the plant life, including seed germination, root hair development, flower senescence, and fruit ripening (Sisler and Yang, 1984). Tomato, as a typical respiratory climacteric fruit, ripening and senescence are largely dependent on endogenous production and ethylene action (Liu et al., 2015). Ethylene is generated from the precursor of methionine through Yang’s cycle. Studies of ethylene biosynthesis have been focused on the isolation and characterization of ACS (1-aminocyclopropane-1-carboxylate synthase) and ACO (1-aminocyclopropane-1-carboxylate oxidase) genes, and ACC (1-aminocyclopropane-1-carboxylate) is the immediate precursor of ethylene (Adams and Yang, 1979; Alexander and Grierson, 2002; Wang et al., 2002). After synthesis, ethylene is perceived by ethylene receptors (ETRs) (Payton et al., 1996). Subsequently, a signaling cascade including both positive and negative regulators modulates a large family of transcription factors called ethylene response factors (ERF) (Thirugnanasambantham et al., 2015). Enormous studies demonstrated that the transcription factor ERF in the AP2/ERF family modulates metabolic pathways involved in fruit ripening and senescence, chlorophyll degradation, fruit softening, and changes in aromas (De Boer et al., 2011; Licausi et al., 2013; Qi et al., 2011). Because ethylene is the key phytohormone regulating fruit ripening, many studies have focused on delaying the action or synthesis of ethylene. For instance, ethylene antagonist 1-methylcyclopropene (1-MCP) was broadly applied to delay fruit ripening and senescence (Song et al., 2018; Watkins et al., 2000). Nitric oxide (NO), an important signaling molecule, was found to delay fruit softening in mango fruit during storage by inhibiting ethylene biosynthesis (Zaharah and Singh, 2011).

Hydrogen sulfide (H2S) is found to be the third most important gasotransmitter in animals and plants after NO and carbon monoxide (CO) (Gadalla and Snyder, 2010; Lisjak et al., 2010; Wang, 2010). H2S participates in almost all aspects of plant life, including seed germination, root organogenesis, stomatal movement, resistance to stress conditions, and plant senescence (Chen et al., 2011; Qiao et al., 2015; Wang et al., 2012; Zhang et al., 2009). Similar to the role of NO in the regulation of fruit ripening and senescence, accumulating studies found that H2S could alleviate postharvest ripening and senescence by modulation of the antioxidant system of kiwifruit, apple, banana, and others (Gao et al., 2013; Ge et al., 2017; Hu et al., 2012). Whether and how H2S is involved in the ripening and senescence of tomato are still unclear. Therefore, in this study, we attempted to illuminate the mechanism of H2S in modulating tomato ripening and senescence. Additionally, the impact of H2S on the ethylene synthesis and signaling pathway in tomato fruit ripening was investigated.

Materials and Methods

Plant materials and sample preparation.

Tomatoes (Solanum lycopersicum cv. Micro Tom) were harvested at the mature green stage; plants were grown in the greenhouse at Hefei University of Technology in Hefei, China. The tomato plants were cultivated at 24 °C with 16-h day and 8-h night conditions, with relative humidity at 50% to 65%. The media for culturing tomato seedlings was composed of nutritional soil, peat soil, and vermiculite in a ratio of 3:5:1. Thirty days post anthesis tomato fruits, which is in the mature-green stage, with similar size and without physical injuries or infections were chosen and stored in containers. The tomatoes were fumigated with ethylene (C2H4) released from 100 mL of 1 g·L−1 ethephon solution or co-treatment of C2H4+H2S released from 150 mL of 1 mmol·L−1 sodium hydrosulfide (NaHS) solution and 100 mL of 1 g·L−1 solution ethephon at 23 ± 0.5 °C with relative humidity of 85% to 90%. After 1 d of storage, the solutions were replaced with distilled water. Each treatment unit comprised six tomatoes, and each experiment was repeated three times. The flesh of fruit without seeds was randomly sampled every other day and stored at −80 °C for subsequent experiments. The changes in tomato fruit color were measured with a color difference meter (model WSC-100; Konica Minolta, Tokyo, Japan), which provided CIE L*, a*, and b* values, where L* indicates lightness, a* indicates chromaticity on a green (−) to red (+) axis, and b* indicates chromaticity on a blue (−) to yellow (+) axis. Each fruit was measured at four equidistant points around the middle area.

Measurement of fruit firmness.

The tomato fruit firmness was measured using a 2-mm-diameter flat probe with a texture analyzer (Model TA XT plus; Stable Micro Systems, Surrey, UK). Fruit firmness value was an average of 10 replicates with standard deviation.

Activity assay of cellulase, polygalacturonase, and pectin lyase.

Frozen tissues (1 g) of tomato were homogenized with 4 mL of 8.8% NaCl containing 10 g·L−1 polyvinyl pyrrolidone (PVPP). The mixture was centrifuged at 10,000 gn for 20 min, and the supernatant was collected for enzyme activity assay.

Cellulase activity was determined as described by Abeles and Biles (1991). One unit (U) of activity was defined as 1 μg of reducing sugar generated per 1 g of fresh weight (FW) per hour. The cellulase activity was expressed as U·g−1 FW.

Polygalacturonase activity was determined according to Pathak and Sanwal (1998). One U of activity was defined as 1 μg of galacturonic acid generated per 1 g of FW per hour. The activity of polygalacturonase was expressed as U·g−1 FW.

Pectin lyase was determined following the method described by Pitt (1988). The reaction was performed in a mixture of 1 mL of 1% pectin solution and 0.5 mL of enzyme extraction at 40 °C for 5 min and ended by adding 3.5 mL of 0.01 mol·L−1 HCl. The absorbance was measured at 235 nm, and 1 U of activity was defined as an absorbance increase of 1.0 per min. The activity of pectin lyase was expressed as U·g−1 FW.

Quantitative reverse-transcription polymerase chain reaction analysis.

Candidate genes encoding cell wall–degrading enzymes, ethylene synthesis, and signaling genes were identified using a hidden Markov model search of protein domains obtained from the Pfam database (http://pfam.xfam.org/) and a local BLASTN analysis of genes (https://solgenomics.net/).

Total RNA from 0.1 g of frozen tomato fruit samples was extracted using the RNA Extraction Kit (Tiangen, Beijing, China). Then, cDNA was synthesized using a reverse-transcription kit (PrimeScript RT Master Mix; Takara, Kyoto, Japan). The cDNA products were used for the quantitative polymerase chain reaction (qPCR) performed using Bio-Rad IQ5 (Hercules, CA) in a 10-μL reaction containing 5 μL of 2 × SYBR Premix Ex Taq (Takara, Kyoto, Japan), 0.8 μL of cDNA, 0.4 μL each for the forward and reverse primers, and 3.4 μL of ddH2O. The specific primers used for the qPCR were designed based on the coding sequence of the genes as shown in the SGN database (https://solgenomics.net/) (Table 1). The qPCR was initiated using a denaturation step at 95 °C for 60 s, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 10 s. Expression of the actin gene in control tomatoes was used for normalization of data. The relative expression levels of all genes were calculated and analyzed by the 2−ΔΔCt method. All analyses were repeated three times using three technical replicates with error bars.

Table 1.

Primers used for the quantitative polymerase chain reaction.

Table 1.

Statistical analysis.

Data were based on three replicates in each experiment, and the experiments were repeated independently three times. Statistical significance was tested using a one-way analysis of variance with IBM SPSS Statistics (SPSS version 20.0; Armonk, NY), and the results were expressed as the means ± sd. Significant differences were calculated following a significance (P < 0.01 or P < 0.05) t test. The Pearson correlation coefficient (R) was used to show the correlation among fruit firmness, cellulase activities, polygalacturonase (PG), pectin lyase (PL), expressions of softening-related genes, ethylene biosynthesis genes, ethylene receptor genes, and ethylene response factors of tomato fruit treated with C2H4 and C2H4+H2S. The heatmap of the parameters was drawn by R script.

Results

Effects of H2S and ethylene on the ripening and senescence of tomatoes during storage.

To evaluate the potential impact of exogenous H2S on fruit ripening, tomatoes at the mature green stage were treated with ethylene or ethylene+H2S for 1 d and subjected to normal conditions. As shown in Fig. 1A, fruits treated with ethylene showed obvious color breaking at 1 d and were fully red at 5 d. However, the ripening process was delayed by C2H4+H2S co-treatment, and tomatoes at the color breaking stage were still observed on day 5. Changes in the skin color of tomato fruits during storage were evaluated via L*, a*, and b*. Figure 1B shows a loss of lightness in both treatment fruits, whereas the L* value of fruits treated with C2H4+H2S decreased more slowly and was maintained at a higher value than that of fruits treated with C2H4. Furthermore, the a* values of fruits treated with C2H4+H2S increased slowly and were significantly lower than those of fruits treated with C2H4 during storage (Fig. 1C), suggesting that the color change from green to red was delayed in tomatoes treated with C2H4+H2S.

Fig. 1.
Fig. 1.

Color changes in tomato fruit treated with C2H4 and C2H4+H2S for 1 d (1 d of storage) and subsequently stored in normal conditions (3, 5, and 7 d of storage) (A). L* indicates lightness (B), a* indicates chromaticity on a green (−) to red (+) axis (C), and b* indicates chromaticity on a blue (−) to yellow (+) axis (D).

Citation: HortScience horts 54, 10; 10.21273/HORTSCI14283-19

Effects of H2S and ethylene on the fruit firmness of tomatoes during storage.

Fruit firmness is an important factor used to evaluate the quality of tomatoes. As shown in Fig. 2A, the firmness of the two groups decreased gradually from day 0 to day 5 during storage. However, ethylene+H2S resulted in significantly higher levels of fruit firmness compared with ethylene treatment during the whole storage period, suggesting that the co-treatment significantly delayed the ethylene-induced softening of tomato fruit.

Fig. 2.
Fig. 2.

Effects of C2H4 and C2H4+H2S treatments on the firmness of tomato fruit (A) and the activities of cell wall–degrading enzymes cellulase (B), polygalacturonase (C), and pectin lyase (D). Fruit were fumigated with C2H4 and C2H4+H2S for 1 d and then stored in normal conditions in a 3-L chamber. Error bars indicate se (n = 10 for fruit firmness and n = 3 for enzyme activities). Asterisks indicate significant differences between fruit treated with C2H4 and C2H4+H2S according to Student’s t test (* P < 0.05; ** P < 0.01).

Citation: HortScience horts 54, 10; 10.21273/HORTSCI14283-19

Effects of H2S and ethylene on the activities of cell wall–degrading enzymes in tomatoes during storage.

To explore the role of H2S in delaying tomato fruit softening, the activities of cell wall–degrading enzymes were determined in fruit. Figure 2B showed the changes of cellulase activities in tomatoes treated with ethylene and ethylene+H2S. Cellulase activity decreased dramatically on day 1, followed by a significant increase. Furthermore, ethylene+H2S treatment induced significantly low levels of cellulase activity on day 3 and day 5 compared with ethylene treatment. As shown in Fig. 2C, the activities of PG decreased gradually with both treatments. However, in comparison with the ethylene treatment, the ethylene+H2S co-treatment apparently maintained lower PG activities from day 1 to day 5. Figure 2D shows that the activities of pectin lyase increased significantly on day 1, followed by a decrease. The activity of the ethylene+H2S co-treatment group of tomatoes on day 1 was 23.1% lower than that of ethylene treatment group of tomatoes. From day 3 to day 5, the pectin lyase activity of tomatoes administered co-treatment was maintained at a significantly lower value (P < 0.01) than that of tomatoes treated with ethylene.

Effects of H2S and ethylene on the expression of softening-related genes in tomatoes.

Fruit softening is attributable to cell wall degradation caused by related enzymes; therefore, the expressions of softening-related genes are modulated during fruit ripening. As shown in Fig. 3A, the transcript level of the pectin lyase gene SlLAT56 increased gradually until day 5 with both treatments during the storage of tomatoes, and no significant difference was observed between ethylene and ethylene+H2S treatment. Figure 3B shows the changes in another pectin lyase gene, SlLAT59, in tomatoes. The expression of SlLAT59 increased slightly on day 1 in tomatoes with both treatments. On day 5 of storage, the transcript of SlLAT59 increased more than two-times that on day 1 with ethylene treatment. Compared with the ethylene group, the expression of SlLAT59 on day 5 with ethylene+H2S co-treatment was significantly lower (P < 0.01) than that with ethylene treatment.

Fig. 3.
Fig. 3.

Relative gene expression patterns of softening-related genes pectin lyases SlLAT56 (A) and SlLAT59 (B) and pectin methylesterase (SlPMEU1) (C) and polygalacturonase (SlPG2) (D) in tomato fruit treated with C2H4 and C2H4+H2S for 1 d. Error bars indicate se (n = 3). Asterisks indicate significant differences between fruit treated with C2H4 and C2H4+H2S according to Student’s t test (* P < 0.05; ** P < 0.01).

Citation: HortScience horts 54, 10; 10.21273/HORTSCI14283-19

Figure 3C shows the changes in the expression of the pectin methylesterase gene SlPMEU1 in tomatoes during storage. The expression of SlPMEU1 decreased gradually with both treatments, but no significant difference was observed between them. As shown in Fig. 3D, the expression of the polygalacturonase gene SlPG2 increased dramatically during tomato storage, and it especially increased on day 5. The gene expression of SlPG2 with ethylene treatment was significantly higher than that with the co-treatment on day 1 and day 5; however, an opposite trend was observed on day 3.

Effects of H2S and ethylene on the expression of ethylene synthesis genes in tomatoes.

Ethylene is the predominant phytohormone that promotes fruit ripening; therefore, the expressions of the genes involved in the biosynthesis of ethylene were analyzed. Figure 4 shows the expression patterns of the six ethylene biosynthesis genes, SlACS2, SlACS3, SlACS6, SlACO1, SlACO3, and SlACO4, in tomatoes treated with ethylene and with ethylene+H2S. As shown in Fig. 4A, the expression of SlACS2 increased and peaked on day 3, followed by a decrease with both treatments. However, ethylene+H2S reduced SlACS2 expression significantly on day 3 compared with ethylene treatment. Figure 4B shows that the expression of SlACS3 fluctuated during tomato storage, and a significantly lower level of SlACS3 expression was observed with co-treatment on day 3 in comparison with ethylene treatment alone. Figure 4C shows that the expression of SlACS6 increased slightly with both treatments and that H2S did not significantly affect the expression. Figure 4D shows that 1 d of fumigation with ethylene induced an increase of SlACO1, followed by a plateau. However, ethylene+H2S treatment sustained significantly lower levels of SlACO1 expression on day 1, followed by a peak on day 3. SlACO1 expression with co-treatment was significantly lower on day 1 and day 5, but it was higher on day 3 compared with ethylene treatment alone. Figure 4E shows that ethylene treatment exhibited a continuous increase in SlACO3 expression, whereas its expression with co-treatment increased slightly and peaked on day 3, followed by a decrease. Ethylene+H2S induced a significantly lower level of SlACO3 expression on day 1 and day 5 during tomato storage compared with ethylene treatment. Changes in patterns similar to those of SlACO1 were observed for SlACO4 expression during tomato storage. Ethylene induced an increase in SlACO4 expression on day 1, but the increase was greatly attenuated with ethylene+H2S cotreatment. The expression of SlACO4 with cotreatment was significantly higher on day 3 and lower on day 5 compared with that with ethylene treatment. H2S repressed the expression of ethylene synthesis genes SlACS2, SlACS3, SlACO1, SlACO3, and SlACO4 in tomatoes during storage to different extents.

Fig. 4.
Fig. 4.

Changes in the gene expression of ethylene biosynthesis–related genes of 1-aminocyclopropane-1-carboxylate synthase [SlACS2 (A), SlACS3 (B), and SlACS6 (C)] and 1-aminocyclopropane-1-carboxylate oxidase [SlACO1 (D), SlACO3 (E), and SlACO4 (F)] in tomato fruit during storage after C2H4 and C2H4+H2S treatment for 1 d. Error bars indicate se (n = 3). Asterisks indicate significant differences between C2H4 and C2H4+H2S co-treated fruit according to Student’s t test (* P < 0.05; ** P < 0.01).

Citation: HortScience horts 54, 10; 10.21273/HORTSCI14283-19

Effects of H2S and ethylene on the expression of ethylene signaling genes in tomatoes.

To evaluate the role of H2S in ethylene signaling, the ethylene signaling genes including ethylene receptors and ethylene response factors were assayed at expression levels. As shown in Fig. 5A, the gene expression of SlETR1 increased slightly with both ethylene and ethylene+H2S treatment in tomatoes during storage, whereas the co-treatment induced a significantly lower expression of SlETR1 on day 5 compared with ethylene alone. Similarly, the expression of SlETR2 increased gradually with both treatments during storage, and H2S was found to attenuate the expression on day 5. Figure 5C shows that SlETR3 increased with ethylene treatment on day 1, followed by a fluctuation. In addition, a significantly lower level of SlETR3 expression with co-treatment compared with ethylene alone was observed on day 1 and day 5, but the trend was reversed on day 3. Figure 5D shows that SlETR4 expression increased gradually with both treatments; however, a decrease was observed on day 5 with co-treatment. Furthermore, ethylene+H2S treatment induced a higher expression of SlETR4 on day 3 compared with ethylene treatment. Figure 5E depicts a trend of increasing expression that was observed with both ethylene and ethylene+H2S treatment; however, ethylene+H2S maintained a significantly lower expression level during the entire storage time compared with control. Figure 5F shows that ethylene treatment induced an increase of SlETR6 on day 1, followed by a plateau, whereas ethylene+H2S treatment attenuated the expression on day 1 and day 5. Ethylene+H2S treatment was found to differentially affect the expression of ethylene receptor genes, and the expression levels of SlETR1, SlETR2, SlETR3, SlETR5, and SlETR6 were significantly downregulated at day 5 in the cotreatment group.

Fig. 5.
Fig. 5.

Changes in the gene expression of ethylene receptor–related genes SlETR1 (A), SlETR2 (B), SlETR3 (C), SlETR4 (D), SlETR5 (E), and SlETR6 (F) in tomato fruit during storage after C2H4 and C2H4+H2S treatment for 1 d. Error bars indicate se (n = 3). Asterisks indicate significant differences between fruit treated with C2H4 and C2H4+H2S according to Student’s t test (* P < 0.05; ** P < 0.01).

Citation: HortScience horts 54, 10; 10.21273/HORTSCI14283-19

Many ethylene response factors become involved in fruit ripening after sensing the signal of ethylene. As shown in Fig. 6A, ethylene treatment induced an increased expression of the ethylene responsive transcription factor SlCRF2 on day 1, followed by a plateau, whereas ethylene+H2S treatment significantly inhibited the expression on day 1 and day 3. Figure 6B shows that SlERF1 increased slightly with both treatments; however, no significant difference was observed. Generally, the trend of increased expression of SlERF2D was observed with both treatments, but H2S caused no significant changes in the expression (Fig. 6C). Figure 6D shows that SlERF2 changed little during storage with both treatments, but ethylene+H2S treatment maintained a significantly lower level of expression during the whole storage time.

Fig. 6.
Fig. 6.

Changes in the gene expression of ethylene response factors SlCRF2 (A), SlERF1 (B), SlERFD2 (C), and SlERF2 (D) in tomato fruit during storage after C2H4 and C2H4+H2S treatment for 1 d. Error bars indicate se (n = 3). Asterisks indicate significant differences between C2H4 and C2H4+H2S cotreated fruit according to Student’s t test (* P < 0.05; ** P < 0.01).

Citation: HortScience horts 54, 10; 10.21273/HORTSCI14283-19

Correlation analysis and heatmap of the values determined in tomatoes during storage.

To determine the factors contributing to tomato fruit softening, the correlation among the value of firmness, activities of cellulase, polygalacturonase, pectin lyase, expressions of softening-related genes (SlLAT56, SlLAT59, SlPMEU1, and SlPG2), ethylene biosynthesis genes (SlACS2, SlACS3, SlACS6, SlACO1, SlACO3, and SlACO4), ethylene receptor genes (SlETR1, SlETR2, SlETR3, SlETR4, SlETR5, and SlETR6), and ethylene response factors (SlCRF2, SlERF1, SlERFD2, SlERF2) were analyzed. As shown in Fig. 7, the data indicated that fruit firmness was negatively correlated with the expression of ethylene-related genes SlACOs, SlACSs, SlETRs, and SlERFs in tomato fruits. Similarly, tomato fruit firmness was negatively correlated with the expression of softening-related genes SlLAT56, SlLAT59, and SlPG2.

Fig. 7.
Fig. 7.

Correlation analysis among the value of firmness, activities of cellulase, polygalacturonase (PG), pectin lyase (PL), expressions of softening-related genes (SlLAT56, SlLAT59, SlPMEU1, and SlPG2), ethylene biosynthesis genes (SlACS2, SlACS3, SlACS6, SlACO1, SlACO3, and SlACO4), ethylene receptor genes (SlETR1, SlETR2, SlETR3, SlETR4, SlETR5, and SlETR6), and ethylene response factors (SlCRF2, SlERF1, SlERFD2, SlERF2) in tomato fruit treated with C2H4 and C2H4+H2S. Pearson’s correlation coefficient among data were analyzed using R scripts.

Citation: HortScience horts 54, 10; 10.21273/HORTSCI14283-19

The heatmap of the physiological parameters of tomatoes during storage was drawn (Fig. 8) to show the difference between ethylene and ethylene+H2S treatment. It could be observed that ethylene+H2S alleviated the softening of tomatoes by attenuating the activities of cell wall–degrading enzymes and their expression, and also the expression of ethylene biosynthesis and signaling genes.

Fig. 8.
Fig. 8.

Heatmap based on the value of firmness, activities of cellulase, polygalacturonase (PG), pectin lyase (PL), expressions of softening-related genes (SlLAT56, SlLAT59, SlPMEU1, and SlPG2), ethylene biosynthesis genes (SlACS2, SlACS3, SlACS6, SlACO1, SlACO3, and SlACO4), ethylene receptor genes (SlETR1, SlETR2, SlETR3, SlETR4, SlETR5, and SlETR6), and ethylene response factors (SlCRF2, SlERF1, SlERFD2, SlERF2) in tomato fruit treated with C2H4 and C2H4+H2S. Co. indicates treatment with C2H4+H2S. Heatmap scale bars are shown in the right part of the figure. Pearson’s correlation coefficient among data were analyzed using R scripts.

Citation: HortScience horts 54, 10; 10.21273/HORTSCI14283-19

Discussion

Fruit ripening is a complex developmental process that is coordinated with the activities of softening-related enzymes and the upregulation of a large set of ripening-related genes. It has been reported that fruit softening is a significant characteristic of fruit ripeness and an important factor affecting tomato fruit quality, storage, and shelf life (Oms-Oliu et al., 2011). Our results showed that the treatment of tomato fruit with exogenous H2S significantly delayed the fruit softening process and inhibited the activity of softening enzymes. The transition of tomato color from green to red was delayed by H2S compared with ethylene alone. Tomatoes were fumigated with H2S released from 1 mm of NaHS or NaHS+ethylene for 1 d and subjected to normal conditions. According to our previous studies, the NaHS concentration used in the present research was within the safety range.

Many enzymes are associated with softening in fruits, including pectinmethylesterase, polygalacturonase, cellulase, and others (Brummell and Harpster, 2001). Polygalacturonase and cellulase are required for the modification of pectin and cellulase, respectively. To alleviate fruit softening, various postharvest measures have been applied, such as ultraviolet-C illumination (Bu et al., 2013) and melatonin treatment (Zhai et al., 2018). In the present study, we found that exogenous H2S alleviated ethylene-induced fruit softening. Furthermore, the enzymes required for cell wall–degrading cellulase, polygalacturonase, and pectin lyase were found to be attenuated by H2S fumigation. Gene expression data consistently showed that the genes encoding pectin lyases (SlLAT56 and SlLAT59), pectin methylesterase (SlPMEU1), and polygalacturonase (SlPG2) were downregulated in tomato fruit treated with H2S for 1 d. Among the enzymes, PG showed very high activity during tomato fruit softening, suggesting that PG might be the key enzyme for cell wall degradation (Brummell and Labavitch, 1997). It was found that the gene SlPG2 was significantly upregulated at a late stage of storage, whereas H2S inhibited the increase (Fig. 3D). The results implied that exogenous H2S alleviated tomato fruit ripening by attenuating the activities of cell wall degradation enzymes and their gene expressions.

Tomato is typically a climacteric fruit, and its ripening and senescence are accompanied by the synthesis of large amounts of ethylene. Therefore, the control of ethylene biosynthesis or its signaling pathway can be applied to alleviate tomato fruit ripening and senescence. For instance, 1-MCP, an ethylene receptor inhibitor, is broadly used during postharvest fruit storage (Song et al., 2018; Watkins et al., 2000), and acetylsalicylic acid shows inhibitory effects on ethylene biosynthesis and signaling during kiwifruit ripening (Yin et al., 2013). In the present research, we studied the regulatory role of exogenous H2S on the expression of ethylene synthesis and signal transduction–related genes in tomatoes. ACC synthase and ACC oxidase are the enzymes that catalyze the conversion of AdoMet to ACC and ACC to ethylene, respectively. It was found that H2S significantly reduced the expression of SlACS2 and SlACS3, whereas SlACS4 genes remained unchanged compared with ethylene. Ethylene significantly upregulates the expression of SlACO1, SlACO3, and SlACO4 after 1 d of treatment, whereas H2S attenuated the increase, suggesting that H2S might inhibit ethylene synthesis at the expression level. These results were consistent with those of our previous report of the role of H2S in alleviating the ripening and senescence of kiwifruit and banana (Ge et al., 2017; Li et al., 2017). The expression patterns of ethylene receptor genes showed that H2S treatment differentially affected their expressions, and expression levels of SlETR1, SlETR2, SlETR3, SlETR5, and SlETR6 were significantly downregulated by H2S. After being recognized by ethylene receptors and a signaling cascade, ethylene has physiological roles in a large family of ERFs, subsequently inducing changes in the metabolism involved in fruit ripening and senescence (Bapat et al., 2010). The present results showed that SlCRF2 and SlERF2 were downregulated by H2S treatment. Exogenous H2S was found to antagonize the synthesis and signaling of ethylene, thereby alleviating fruit ripening in tomatoes.

Texture softening is a predominant characteristic of ethylene-induced fruit ripening. We found that H2S could attenuate the expression of ethylene signaling and pathway genes. The cell wall degradation enzymes (CWDE) was also decreased by H2S treatment. Therefore, the correlation was analyzed and the results showed that fruit firmness was negative correlated with the expression of ethylene-related genes SlACOs, SlACSs, SlETRs, and SlERFs in tomato fruits. It is difficult to say whether H2S regulates these two events separately or in a linear way. Accumulating reports found that ethylene can act upstream of CWDE. For instance, the expression of CWDE is ethylene-dependent; therefore, fruit softening and cell wall disassembly are regulated by ethylene in Charentais melon (Nishiyama et al., 2007). The ERF DkERFs in persimmon fruit was found to directly bind to the promoter of a CWDE gene DkXTH9 and regulate its gene expression (Wang et al., 2017). Therefore, the role of H2S in regulating fruit softening could be as a linear signaling pathway (H2S-ethylene-CWDE).

Short-term application of H2S alleviates fruit softening by inhibiting the activities and gene expressions of cell wall–degrading enzymes. This study also demonstrated that H2S downregulates ethylene biosynthesis and signaling genes, effectively protecting cell wall components from being degraded. This work provides insight into the multifaceted role of H2S in fruit senescence and implicates the potential application of H2S during postharvest fruit storage.

Literature Cited

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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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Contributor Notes

Funding for this work was provided by the National Natural Science Foundation of China (31670278, 31970200, 31970312, 31901993, 31872078), the Anhui Provincial Science and Technology Major Project (16030701073), the Earmarked Fund for the China Agriculture Research System (CARS-10-B1), the Fundamental Research Funds for the Central Universities (JZ2018HGTB0241, JZ2018HGBZ0160), the Natural Science Foundations of Anhui Province (1908085MC72), the Key Research and Development Program of Anhui Province (201904a06020031), and National Undergraduate Training Programs for Innovation of China (No. 201810359054, 2018CXCYS195).

K.-D.H., X.-Y.Z., and S.-S.W. contributed equally to this work.

G.-F.Y. and H.Z. are the corresponding authors. E-mail: yaogaifang@hfut.edu.cn or hzhanglab@hfut.edu.cn.

  • View in gallery

    Color changes in tomato fruit treated with C2H4 and C2H4+H2S for 1 d (1 d of storage) and subsequently stored in normal conditions (3, 5, and 7 d of storage) (A). L* indicates lightness (B), a* indicates chromaticity on a green (−) to red (+) axis (C), and b* indicates chromaticity on a blue (−) to yellow (+) axis (D).

  • View in gallery

    Effects of C2H4 and C2H4+H2S treatments on the firmness of tomato fruit (A) and the activities of cell wall–degrading enzymes cellulase (B), polygalacturonase (C), and pectin lyase (D). Fruit were fumigated with C2H4 and C2H4+H2S for 1 d and then stored in normal conditions in a 3-L chamber. Error bars indicate se (n = 10 for fruit firmness and n = 3 for enzyme activities). Asterisks indicate significant differences between fruit treated with C2H4 and C2H4+H2S according to Student’s t test (* P < 0.05; ** P < 0.01).

  • View in gallery

    Relative gene expression patterns of softening-related genes pectin lyases SlLAT56 (A) and SlLAT59 (B) and pectin methylesterase (SlPMEU1) (C) and polygalacturonase (SlPG2) (D) in tomato fruit treated with C2H4 and C2H4+H2S for 1 d. Error bars indicate se (n = 3). Asterisks indicate significant differences between fruit treated with C2H4 and C2H4+H2S according to Student’s t test (* P < 0.05; ** P < 0.01).

  • View in gallery

    Changes in the gene expression of ethylene biosynthesis–related genes of 1-aminocyclopropane-1-carboxylate synthase [SlACS2 (A), SlACS3 (B), and SlACS6 (C)] and 1-aminocyclopropane-1-carboxylate oxidase [SlACO1 (D), SlACO3 (E), and SlACO4 (F)] in tomato fruit during storage after C2H4 and C2H4+H2S treatment for 1 d. Error bars indicate se (n = 3). Asterisks indicate significant differences between C2H4 and C2H4+H2S co-treated fruit according to Student’s t test (* P < 0.05; ** P < 0.01).

  • View in gallery

    Changes in the gene expression of ethylene receptor–related genes SlETR1 (A), SlETR2 (B), SlETR3 (C), SlETR4 (D), SlETR5 (E), and SlETR6 (F) in tomato fruit during storage after C2H4 and C2H4+H2S treatment for 1 d. Error bars indicate se (n = 3). Asterisks indicate significant differences between fruit treated with C2H4 and C2H4+H2S according to Student’s t test (* P < 0.05; ** P < 0.01).

  • View in gallery

    Changes in the gene expression of ethylene response factors SlCRF2 (A), SlERF1 (B), SlERFD2 (C), and SlERF2 (D) in tomato fruit during storage after C2H4 and C2H4+H2S treatment for 1 d. Error bars indicate se (n = 3). Asterisks indicate significant differences between C2H4 and C2H4+H2S cotreated fruit according to Student’s t test (* P < 0.05; ** P < 0.01).

  • View in gallery

    Correlation analysis among the value of firmness, activities of cellulase, polygalacturonase (PG), pectin lyase (PL), expressions of softening-related genes (SlLAT56, SlLAT59, SlPMEU1, and SlPG2), ethylene biosynthesis genes (SlACS2, SlACS3, SlACS6, SlACO1, SlACO3, and SlACO4), ethylene receptor genes (SlETR1, SlETR2, SlETR3, SlETR4, SlETR5, and SlETR6), and ethylene response factors (SlCRF2, SlERF1, SlERFD2, SlERF2) in tomato fruit treated with C2H4 and C2H4+H2S. Pearson’s correlation coefficient among data were analyzed using R scripts.

  • View in gallery

    Heatmap based on the value of firmness, activities of cellulase, polygalacturonase (PG), pectin lyase (PL), expressions of softening-related genes (SlLAT56, SlLAT59, SlPMEU1, and SlPG2), ethylene biosynthesis genes (SlACS2, SlACS3, SlACS6, SlACO1, SlACO3, and SlACO4), ethylene receptor genes (SlETR1, SlETR2, SlETR3, SlETR4, SlETR5, and SlETR6), and ethylene response factors (SlCRF2, SlERF1, SlERFD2, SlERF2) in tomato fruit treated with C2H4 and C2H4+H2S. Co. indicates treatment with C2H4+H2S. Heatmap scale bars are shown in the right part of the figure. Pearson’s correlation coefficient among data were analyzed using R scripts.

  • Abeles, F.B. & Biles, C.L. 1991 Cellulase activity in developing apple fruits Scientia Hort. 47 77 87

  • Adams, D.O. & Yang, S.F. 1979 Ethylene biosynthesis: Identification of 1-aminocyclopropane-l-carboxylic acid as an intermediate in the conversion of methionine to ethylene Proc. Natl. Acad. Sci. USA 76 170 174

    • Search Google Scholar
    • Export Citation
  • Alexander, L. & Grierson, D. 2002 Ethylene biosynthesis and action in tomato: A model for climacteric fruit ripening J. Expt. Bot. 53 2039 2055

  • Bapat, V.A., Trivedi, P.K., Ghosh, A., Sane, V.A., Ganapathi, T.R. & Nath, P. 2010 Ripening of fleshy fruit: Molecular insight and the role of ethylene Biotechnol. Adv. 28 94 107

    • Search Google Scholar
    • Export Citation
  • Beecher, G.R. 1998 Nutrient content of tomatoes and tomato products Proc. Soc. Exp. Biol. Med. 218 98 100

  • Brummell, D.A. & Harpster, M.H. 2001 Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants Plant Mol. Biol. 47 311 340

    • Search Google Scholar
    • Export Citation
  • Brummell, D.A. & Labavitch, J.M. 1997 Effect of antisense suppression of endopolygalacturonase activity on polyuronide molecular weight in ripening tomato fruit and in fruit homogenates Plant Physiol. 115 717 725

    • Search Google Scholar
    • Export Citation
  • Bu, J., Yu, Y., Aisikaer, G. & Ying, T. 2013 Postharvest UV-C irradiation inhibits the production of ethylene and the activity of cell wall-degrading enzymes during softening of tomato (Lycopersicon esculentum L.) fruit Postharvest Biol. Technol. 86 337 345

    • Search Google Scholar
    • Export Citation
  • Chen, J., Wu, F.H., Wang, W.H., Zheng, C.J., Lin, G.H., Dong, X.J., He, J.X., Pei, Z.M. & Zheng, H.L. 2011 Hydrogen sulphide enhances photosynthesis through promoting chloroplast biogenesis, photosynthetic enzyme expression, and thiol redox modification in Spinacia oleracea seedlings J. Expt. Bot. 62 4481 4493

    • Search Google Scholar
    • Export Citation
  • De Boer, K., Tilleman, S., Pauwels, L., Vanden Bossche, R., De Sutter, V., Vanderhaeghen, R., Hilson, P., Hamill, J.D. & Goossens, A. 2011 APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis Plant J. 66 1053 1065

    • Search Google Scholar
    • Export Citation
  • Fischer, R.L. & Bennett, A.B. 1991 Role of cell wall hydrolases in fruit ripening Annu. Rev. Plant Physiol. Plant Mol. Biol. 42 675 703

  • Gadalla, M.M. & Snyder, S.H. 2010 Hydrogen sulfide as a gasotransmitter J. Neurochem. 113 14 26

  • Gao, S.P., Hu, K.D., Hu, L.Y., Li, Y.H., Han, Y., Wang, H.L., Lv, K., Liu, Y.S. & Zhang, H. 2013 Hydrogen sulfide delays postharvest senescence and plays an antioxidative role in fresh-cut kiwifruit HortScience 48 1385 1392

    • Search Google Scholar
    • Export Citation
  • Ge, Y., Hu, K.D., Wang, S.S., Hu, L.Y., Chen, X.Y., Li, Y.H., Yang, Y., Yang, F. & Zhang, H. 2017 Hydrogen sulfide alleviates postharvest ripening and senescence of banana by antagonizing the effect of ethylene PLoS One 12 E0180113

    • Search Google Scholar
    • Export Citation
  • Giovannoni, J.J. 2007 Fruit ripening mutants yield insights into ripening control Curr. Opin. Plant Biol. 10 283 289

  • Grierson, D., Maunders, M.J., Slater, A., Ray, J., Bird, C.R., Schuch, W., Holdsworth, M.J., Tucker, G.A. & Knapp, J.E. 1986 Gene expression during tomato ripening Philos. Trans. R. Soc. Lond. B Biol. Sci. 314 399 410

    • Search Google Scholar
    • Export Citation
  • Gwanpua, S.G., Mellidou, I., Boeckx, J., Kyomugasho, C., Bessemans, N., Verlinden, B.E., Hertog, M.L.A.T.M., Hendrickx, M., Nicolai, B.M. & Geeraerd, A.H. 2016 Expression analysis of candidate cell wall-related genes associated with changes in pectin biochemistry during postharvest apple softening Postharvest Biol. Technol. 112 176 185

    • Search Google Scholar
    • Export Citation
  • Hu, L.Y., Hu, S.L., Wu, J., Li, Y.H., Zheng, J.L., Wei, Z.J., Liu, J., Wang, H.L., Liu, Y.S. & Zhang, H. 2012 Hydrogen sulfide prolongs postharvest shelf life of strawberry and plays an antioxidative role in fruits J. Agr. Food Chem. 60 8684 8693

    • Search Google Scholar
    • Export Citation
  • Li, T.T., Li, Z.R., Hu, K.D., Hu, L.Y., Chen, X.Y., Li, Y.H., Yang, Y., Yang, F. & Zhang, H. 2017 Hydrogen sulfide alleviates kiwifruit ripening and senescence by antagonizing effect of ethylene HortScience 52 1556 1562

    • Search Google Scholar
    • Export Citation
  • Licausi, F., Ohme-Takagi, M. & Perata, P. 2013 APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors: Mediators of stress responses and developmental programs New Phytol. 199 639 649

    • Search Google Scholar
    • Export Citation
  • Lisjak, M., Srivastava, N., Teklic, T., Civale, L., Lewandowski, K., Wilson, I., Wood, M.E., Whiteman, M. & Hancock, J.T. 2010 A novel hydrogen sulfide donor causes stomatal opening and reduces nitric oxide accumulation Plant Physiol. Biochem. 48 931 935

    • Search Google Scholar
    • Export Citation
  • Liu, M., Pirrello, J., Chervin, C., Roustan, J.P. & Bouzayen, M. 2015 Ethylene control of fruit ripening: Revisiting the complex network of transcriptional regulation Plant Physiol. 169 2380 2390

    • Search Google Scholar
    • Export Citation
  • Musse, M., Quellec, S., Cambert, M., Devaux, M.F., Lahaye, M. & Mariette, F. 2009 Monitoring the postharvest ripening of tomato fruit using quantitative MRI and NMR relaxometry Postharvest Biol. Technol. 53 22 35

    • Search Google Scholar
    • Export Citation
  • Nishiyama, K., Guis, M., Rose, J.K., Kubo, Y., Bennett, K.A., Wangjin, L., Kato, K., Ushijima, K., Nakano, R., Inaba, A., Bouzayen, M., Latche, A., Pech, J.C. & Bennett, A.B. 2007 Ethylene regulation of fruit softening and cell wall disassembly in Charentais melon J. Expt. Bot. 58 1281 1290

    • Search Google Scholar
    • Export Citation
  • Oms-Oliu, G., Hertog, M.L.A.T.M., Van de. Poel, B., Ampofo-Asiama, J., Geeraerd, A.H. & Nicolaï, B.M. 2011 Metabolic characterization of tomato fruit during preharvest development, ripening, and postharvest shelf-life Postharvest Biol. Technol. 62 7 16

    • Search Google Scholar
    • Export Citation
  • Pathak, N. & Sanwal, G.G. 1998 Multiple forms of polygalacturonase from banana fruits Phytochemistry 48 249 255

  • Payton, S., Fray, R.G., Brown, S. & Grierson, D. 1996 Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission Plant Mol. Biol. 31 1227 1231

    • Search Google Scholar
    • Export Citation
  • Pitt, D. 1988 Pectin lyase from Phoma medicaginis var. pinodella Methods Enzymol. 161 350 354

  • Qi, W., Sun, F., Wang, Q., Chen, M., Huang, Y., Feng, Y.Q., Luo, X. & Yang, J. 2011 Rice ethylene-response AP2/ERF factor OsEATB restricts internode elongation by down-regulating a gibberellin biosynthetic gene Plant Physiol. 157 216 228

    • Search Google Scholar
    • Export Citation
  • Qiao, Z., Tao, J., Liu, Z., Zhang, L., Jin, Z., Liu, D. & Pei, Y. 2015 H2S acting as a downstream signaling molecule of SA regulates Cd tolerance in Arabidopsis Plant Soil 393 137 146

    • Search Google Scholar
    • Export Citation
  • Sisler, E.C. & Yang, S.F. 1984 Ethylene, the gaseous plant hormone Bioscience 34 234 238

  • Song, J.S., Yoo, S.K. & Kim, D.S. 2018 The effect of 1-(3-phenyl-propyl)cyclopropene versus 1-MCP on the quality and storage life of tomato (Solanum lycopersicum) fruit Postharvest Biol. Technol. 145 20 26

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