Methyl jasmonate-dependent hydrogen sulfide mediates melatonin-induced aphid resistance of watermelon

Abstract

As one of the most destructive pests, aphids cause significant damage on various agricultural and horticultural crops. Recently, melatonin has been shown to enhance plant resistance to aphids; however, the underlying mechanisms remain unclear. In this study, our results showed that melatonin, MeJA, and H₂S enhanced aphid resistance of watermelon in a dose-dependent manner, accompanied by increases in the defense-related enzyme activities and lignin accumulation. On the plants pretreated with 100 μM melatonin, 100 μM MeJA, and 50 μM NaHS, the numbers of aphids were 86.0%, 59.6%, and 47.9% lower than that on control plants, respectively, after aphid infestation for 7 days. Melatonin application induced MeJA and H₂S accumulation in response to aphid infestation, while inhibition of MeJA and H₂S accumulation attenuated or abolished melatonin-induced defense response and aphid resistance, suggestive of the involvement of MeJA and H₂S in melatonin-induced aphid resistance. MeJA also increased H₂S accumulation, but inhibition of MeJA biosynthesis prevented melatonin-enhanced H₂S accumulation, suggesting that MeJA mediates melatonin-induced H₂S accumulation. Furthermore, inhibition of H₂S production attenuated MeJA-induced defense response and aphid resistance. Taken together, the current study reveals a novel mechanism in which MeJA-dependent H₂S signaling is involved in melatonin-induced defense response and subsequent aphid resistance. The increasing concern to minimize the use of pesticides and to switch onto sustainable and natural control strategies indicates the great exploitation of such a mechanism in combating aphids.

INTRODUCTION

Aphids (Hemiptera: Aphidoidea) are small soft-bodied insects that have a wide host range and are specialized to feed from the plant phloem (Aheer et al., 2008). Among the more than 4000 known aphid species, around 250 are among the most destructive pests, because of their parthenogenesis, rapid reproduction, extensive crop damage, transmission of more than 200 plant pathogenic viruses, and resistance to a mass of insecticides (Brault et al., 2010; Dedryver et al., 2010; Nalam et al., 2019). For some crops, virus by aphid transmission often does more harm than aphid feeding (Züst & Agrawal, 2016). In addition, honeydew, a sticky substance excreted by aphids, results in the growth of sooty molds on leaf surfaces and consequently inhibits plant photosynthesis (Wilkinson et al., 1997).

To cope with aphid infestation, plants have evolved a range of unique mechanisms of recognition, signaling, and defenses (Chen, 2008; Züst & Agrawal, 2016). Plant can defend against aphids by interfering with aphid growth, survival, and fecundity or impacting aphid feeding behavior, such as plant choice (Nalam et al., 2019). For antibiosis mechanism, plant receptors recognize aphid feeding via elicitors and initiate defense signaling, such as phytohormonal signaling and Ca²⁺ signaling, inducing phloem sealing, systemic response, increase in epicuticular wax and trichomes on plant surface, and biosynthesis of defensive plant secondary metabolites and protease inhibitors (Nalam et al., 2019; Züst & Agrawal, 2016).

Melatonin (N-acetyl-5-methoxy-tryptamine) is a universal and pleiotropic molecule with many diverse actions in animal, plant, and many other kingdoms (Arnao, 2014; Dubbels et al., 1995; Hardeland et al., 2011). Since the discovery of melatonin in vascular plants in 1995 (Dubbels et al., 1995; Hattori et al., 1995), melatonin has been widely reported as a critical and pleiotropic regulatory molecule in plant defending against various abiotic stresses and microbial infections (fungi, bacteria, and viruses) (Arnao & Hernandez-Ruiz, 2014; Gao et al., 2022; Zhang et al., 2014). However, the role and mechanism of melatonin in regulating plant response to insect pests has rarely been reported. One study carried out in pea aphid (Acyrthosiphon pisum) revealed that feeding with high concentration of melatonin can significantly increase mortality before the adult stage, delay adult development, and decrease fecundity and such effect of melatonin increased with the increase concentration (Gao & Hardie, 1997). The latest research demonstrates that exogenous melatonin can enhance tobacco resistance against aphid by promoting the development of glandular trichomes and consequent increase in cembranoid production (Song et al., 2022). It is necessary to further explore the signaling transduction in melatonin regulating aphid resistance of plants.

Increasing studies have proven that phytohormones play central roles in signaling transduction and activation of defense systems during plant response to aphid feeding (Züst & Agrawal, 2016). Previous chewing damage of leaf by insects triggers JA signaling and consequently negatively affects aphid performance (Cooper et al., 2004). Exogenous application of JA or its derivative (e.g., methyl jasmonate, MeJA) inhibited aphid population growth on various plant species such as soybean, tomato, and cucumber (Ali & Agrawal, 2014; Qi et al., 2020; Selig et al., 2016). Moreover, overexpression and suppression of lipoxygenase (LOX) 2.2, a key JA biosynthesis-related gene, reduces and increases aphid fecundity, respectively, in barley (Losvik et al., 2017). However, the mechanism by which JA activates defense response against aphid remains unclear.

Besides phytohormones, many secondary messengers, such as calcium (Ca²⁺), reactive oxygen species (ROS), nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H₂S), also play critical role in regulating plant defense responses to environmental stresses. H₂S is considered as the third gaseous messenger, after NO CO (Wang, 2002). In the past few years, increasing evidence has shown that H₂S plays multiple roles in regulating a wide range of physiological processes (e.g., seed germination, root organogenesis, photosynthesis, stomatal movement, fruit ripening, and leaf senescence) and plant responses to various abiotic stresses (e.g., extreme temperatures, excess copper, salinity, and drought) and microbial pathogens (Choudhary et al., 2022; Yang et al., 2022). However, little is known about the involvement of H₂S in plant defensing against insect pests.

Watermelon is a widely cultivated vegetable crop species worldwide and is particularly vulnerable to melon-cotton aphid (Aphis gossypii Glover), a widely distributed species that damages hundreds of crops such as melons and cotton (Blackman & Eastop, 2007). Increasing evidence indicates that melatonin confers plant tolerance to some abiotic stresses via interacting with MeJA and hydrogen sulfide H₂S (Li, Guo et al., 2021; Vojtovi et al., 2021). These findings raise a possible cross talk between melatonin and MeJA or H₂S in plant response to aphid attack. To test this assumption, we demonstrated the roles of melatonin, MeJA, and H₂S and their cross talk in regulating aphid resistance of watermelon. The present study provides a novel insight into understanding the regulation mechanism of melatonin-mediated aphid resistance of plants.

MATERIALS AND METHODS Plant materials and aphid infestation

Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai cv. Nongkeda No. 5] was used in this study. Germinated seeds were sown into plastic pots filled with commercial peat-based compost. The seedlings were grown in a greenhouse at Northwest A&F University with a temperature ranging from 18.0°C to 36.0°C, light intensity of 500 μmol m⁻² s⁻¹, and relative humidity of 65%–80%. Plants were watered every 2 days and fertilized with Hoagland's nutrient solution every 3 days.

Aphids (Aphis gossypii Glover) were cultured as described previously (Liang et al., 2016). An aphid colony was collected from watermelon plants grown in a greenhouse at Northwest A&F University and reared on watermelon seedlings at 25/18°C (day/night), 500 μmol m⁻² s⁻¹ light intensity, 50%–70% relative humidity, and a 14-h–10-h light–dark photoperiod. The offspring of this aphid colony were used to infest watermelon seedlings.

Watermelon seedlings with four true leaves were used for aphid infestation. After 4-h starvation period, six adult apterous (wingless) aphids were transferred onto the lower surface of the third leaf from the bottom per seedling. Noninfested seedlings were used as the control. Each seedling was covered with a cylindrical device, which consists of insect-proof net (100 mesh) at the top and transparent film at the bottom, to prevent aphids from moving between plants.

Experimental design

To investigate the effects of exogenous application of melatonin, MeJA, and H₂S on the aphid resistance of watermelon seedlings, the leaves were sprayed with melatonin (0, 50, 100, 200, 400, or 600 μM) or MeJA (0, 50, 100, 200, or 400 μM), or the roots were treated with NaHS (0, 20, 50, 100, 300, or 500 μM) for two times at 1-day interval. NaHS is the most widely used donor that directly and instantaneously releases H₂S in plants (Huang et al., 2021). Melatonin and MeJA solutions were prepared as described previously (Li, Guo et al., 2021). Twelve hours later, the third true leaf from the bottom was infested with six adult apterous (wingless) aphids. The number of aphids on individual plants was counted on the 3rd and 7th day after infestation. Each experiment was repeated three times and each treatment included at least 15 seedlings.

To assess the involvement of MeJA and H₂S and their interactions in melatonin-induced aphid resistance, diethyldithiocarbamic acid (DIECA, a JA biosynthesis inhibitor) and hypotaurine (HT, a H₂S scavenger) were used to block MeJA biosynthesis and H₂S accumulation, respectively (Iqbal et al., 2021; Li, Guo et al., 2021). The leaves were pretreated with 5 mM diethyldithiocarbamic acid or the roots were pretreated with 100 μM hypotaurine. Eight hours later, the plants were sprayed with melatonin or MeJA, and 12 h later, they were infested with aphids. Notably, at 6 h after DIECA treatment, the leaves were rinsed with double distilled water to avoid the direct effect of DIECA on aphids.

Assay of defense enzyme activities and lignin content

The protein content was determined according to the method of Bradford and Williams (Bradford & Williams, 1976). The activities of trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) were measured by means of inhibition of esterase activity of trypsin and chymotrypsin, respectively, as described by Casaretto and Corcuera (1998). TI and CI activity was expressed as activity per gram protein. Polyphenol oxidase (PPO) activity was measured as described by Xin et al. (2016). Phenylalanine ammonia lyase (PAL) activity was measured following the method of Dubery and Smit (1994). PPO and PAL activity was expressed as activity per gram fresh weight. For the determination of lignin content, the leaf samples were dried at 80°C to constant weight, pulverized and passed through a 50-mesh sieve. The lignin content was determined using a lignin assay kit (Solarbio) according to the instructions provided by the manufacturer. The lignin content was expressed as the weight percentage of dry weight.

Quantification of melatonin and methyl jasmonate

Melatonin extraction was performed according to the method of Pape and Lüning (2006). Methyl jasmonate (MeJA) extraction was performed as described by Pan et al. (2010). Melatonin and MeJA levels were quantified using their respective ELISA kit (China Agricultural University) following the manufacturer's instructions.

Analysis of hydrogen sulfide

Hydrogen sulfide (H₂S) content was measured as described previously (Li et al., 2018). Frozen leaf samples (0.3 g) were homogenized in 3 mL of phosphate-buffered saline (pH 6.8, 50 mM) containing 0.1 M EDTA and 0.2 M ascorbic acid. Then, the homogenate was transferred to a test tube containing 10 mM L-Cys, 0.1 M phosphate-buffered saline (pH 7.4), and 2 mM phosphor-pyridoxal at room temperature. Released H₂S was trapped in 3 mL of 0.5% (w/v) zinc acetate solution. After reaction for 30 min, 0.3 mL of 20 mM dimethyl-$p$-phenylenediamine dissolved with HCl (7.2 mM) was added to the zinc acetate trap, followed by 1.2 mL of HCl containing 30 mM ferric ammonium sulfate. After incubation at room temperature for 15 min, the content of H₂S was recorded at OD₆₆₇.

Statistical analysis

The experiments were performed in a completely randomized design. Each experiment was repeated three times and each treatment included at least 15 seedlings Each replicate included at least 15 seedlings. Analysis of variance (ANOVA) was performed using SPSS statistics 19 (SPSS Inc.), followed by Tukey's test at p < 0.05.

RESULTS

We first analyzed the effects of melatonin at different concentrations on aphid resistance of watermelon seedlings. Seven days after aphid infestation, the numbers of aphids increased by 27.2-fold in control plants (Figure 1). Exogenous melatonin at appropriate dose (50–600 μM) enhanced watermelon resistance against aphids. The most effective concentration of melatonin was 100 μM. On the plants pretreated with 100 μM melatonin, the numbers of aphids were 72.1% and 86.0% lower than that on control plants, after aphid infestation for 3 and 7 days, respectively. The protective effect of melatonin against aphid was attenuated with melatonin concentrations both lower and higher than 100 μM. Additionally, the endogenous content of melatonin in watermelon leaves increased from 4.8 to 16.9 ng g⁻¹, after pretreatment with 100 μM melatonin for 12 h (Figure S1). Inhibition of melatonin biosynthesis by p-chlorophenylalanine (CPA, a melatonin synthesis inhibitor) (Park, 2011; Wen et al., 2016) decreased the endogenous melatonin content and aphid resistance (Figures S1 and S2).

FIGURE 1. Effects of melatonin on aphid resistance of watermelon. Watermelon seedlings at the four-leaf stage were pretreated with melatonin at 0, 50, 100, 200, 400 or 600 μM for two times at 1-day interval. Twelve hours later, the seedlings were infested with aphids. The number of aphids on individual plants was counted on the 3rd and 7th day after infestation. (a) The number of aphids. (b) Phenotype of leaves after aphid infestation for 7 days. In (a), data are expressed as means ± SDs (n = 15). Means denoted with different letters differ significantly (p [less than] 0.05, Tukey's test).

Lignin and defense-related enzymes, such as trypsin inhibitor (TI), chymotrypsin inhibitor (CI), polyphenol oxidase (PPO), and phenylalanine ammonia lyase (PAL), play critical roles in plants defending against aphids. The activities of TI and PAL were almost unchanged or decreased, but the activities of CI and PPO and the content of lignin were significantly increased by aphid infestation (Figure 2). Importantly, melatonin increased the activities of TI, CI, PPO, and PAL and the content of lignin under both normal and aphid infestation conditions, with some exceptions. For instance, the activities of TI, CI, PPO, and PAL and the content of lignin in melatonin-pretreated plants were 30.3%, 48.8%, 44.7%, 30.3%, and 35.4% higher than that in the control plants, respectively, on the 3rd day after aphid infestation.

FIGURE 2. Activities of defense-related enzymes and the accumulation of lignin in response to melatonin and aphid infestation. The treatments were the same as those described in Figure 1. The activities of defense-related enzymes and the content of lignin were measured on the 3rd and 7th day after the aphid infestation. The data are means of three replicates (±SDs). Means denoted with different letters differ significantly (p [less than] 0.05, Tukey's test). CI, chymotrypsin inhibitor; DW, dry weight; FW, fresh weight; PAL, phenylalanine ammonia lyase; PPO, polyphenol oxidase; TI, trypsin inhibitor.

To examine the role of MeJA and H₂S in melatonin-induced aphid resistance, we first analyzed the effects of exogenous melatonin on the accumulation of MeJA and H₂S. Under normal conditions, the content of MeJA and H₂S fluctuates with growth time (Figure 3). Aphid infestation induced the accumulation of MeJA at 12 h and on the 7th day and the accumulation of H₂S on the 3rd and 7th day. Importantly, plants sprayed with melatonin showed higher levels of MeJA and H₂S than the control plants after aphid infestation. For instance, MeJA and H₂S contents in melatonin-pretreated plants were 24.9% and 19.1% higher than that in the control plants on the 3rd day after aphid infestation.

FIGURE 3. Accumulation of methyl jasmonate (MeJA) and H2S in leaves in response to melatonin and aphid infestation. The leaves were pretreated with 100 μM melatonin for two times at 1-day interval. Twelve hours later, the seedlings were infested with aphids. The contents of MeJA and H2S were determined at indicated times. The data are the means of three replicates (±SDs). FW, fresh weight.

As well as melatonin, application of both MeJA (50–400 μM) and NaHS (20–500 μM) inhibited the increases in aphid populations on watermelon leaves (Figure 4). The most effective concentration of MeJA and NaHS was 100 and 50 μM, respectively. On the plants pretreated with 100 μM MeJA and 50 μM NaHS, the numbers of aphids were 59.6% and 47.9% lower than that on control plants, respectively, after aphid infestation for 7 days. The endogenous contents of MeJA and H₂S increased by 88.4% and 24.6% after 100 μM MeJA and 50 μM NaHS application, respectively (Figure S1). Moreover, MeJA and NaHS pretreatment increased the activities of TI, CI, PPO, and PAL and the content of lignin under both normal and aphid infestation conditions, with some exceptions (Figure 5). For instance, the activities of TI, CI, PPO, and PAL and the content of lignin in MeJA-pretreated plants were 36.6%, 95.9%, 90.4%, 24.7%, and 48.7% higher than that in the control plants, respectively, on the 7th day after aphid infestation.

FIGURE 4. Effects of methyl jasmonate (MeJA) and NaHS on aphid resistance of watermelon. (a, b) Aphid populations and leaf phenotypes of MeJA-pretreated plants after aphid infestation. The leaves were sprayed with MeJA (MJ) at 0, 50, 100, 200, or 400 μ M for two times at 1-day interval. Twelve hours later, the seedlings were infested with aphids. (c, d) Aphid populations and leaf phenotypes of NaHS pretreated plants after aphid infestation. The roots were pretreated with NaHS (NS) at 0, 20, 50, 100, 300, or 500 μ M for two times at 1-day interval. Twelve hours later, the leaves were infested with aphids. In (a, c), data are expressed as means ± SDs (n = 15). Means denoted with different letters differ significantly (p [less than] 0.05, Tukey's test).

FIGURE 5. Effects of (a) methyl jasmonate (MeJA) and (b) NaHS on defense-related enzyme activities and lignin accumulation under normal or aphid infestation conditions. The treatments were the same as those described in Figure 4. The activities of defense-related enzymes and the content of lignin were measured on the 3rd and 7th day after the aphid infestation. Values are the means ± SD of three replicates. Means denoted with different letters differ significantly (p [less than] 0.05, Tukey's test). CI, chymotrypsin inhibitor; DW, dry weight; FW, fresh weight; PAL, phenylalanine ammonia lyase; PPO, polyphenol oxidase; TI, trypsin inhibitor.

Pretreatment with DIECA and HT decreased the endogenous level of MeJA and H₂S, respectively, and increased aphid populations (Figures S1 and S2). Moreover, DIECA and HT application abolished and attenuated the melatonin-induced aphid resistance, respectively, as reflected by the increases in aphid populations (Figure 6). In line with aphid populations, melatonin-induced increases in the activities of TI, CI, PPO, and PAL and the content of lignin were also attenuated or abolished by DIECA and HT application, with some exceptions. These results suggest that both MeJA and H₂S are involved in melatonin-induced aphid resistance and increases in defense-related enzyme activities and lignin accumulation.

FIGURE 6. Involvement of methyl jasmonate (MeJA) and H2S in melatonin-induced aphid resistance and defense response in watermelon plants. The leaves were sprayed with diethyldithiocarbamic acid (DIECA, 5 mM) or the roots were treated with hypotaurine (HT, 100 μM) 8 h prior to melatonin (100 μM) treatment. Twelve hours later, the plants were infested with aphids. On the 3rd and 7th day after the aphid infestation, the number of aphids was counted and the activities of defense-related enzymes and the content of lignin were measured on the 3rd and 7th day after the aphid infestation. Data of aphid number are expressed as means ± SDs (n = 15). Values of enzyme activities and lignin content are the means ± SD of three replicates. Means denoted with different letters differ significantly (p [less than] 0.05, Tukey's test). CI, chymotrypsin inhibitor; DW, dry weight; FW, fresh weight; PAL, phenylalanine ammonia lyase; PPO, polyphenol oxidase; TI, trypsin inhibitor.

To investigate the interaction between MeJA and H₂S in melatonin-mediated aphid resistance, we first analyzed the effects of MeJA on H₂S accumulation. Like melatonin, MeJA pretreatment also induced the accumulation of endogenous H₂S under normal and especially aphid infestation conditions (Figure 7a). However, inhibition of MeJA biosynthesis by DIECA pretreatment completely abolished melatonin-induced increases in the H₂S accumulation after aphid infestation (Figure 7b). These data demonstrate that MeJA mediates melatonin-induced H₂S production in plant response to aphid infestation. To further characterize the involvement of H₂S in MeJA-induced aphid resistance, the roots were treated with HT 8 h prior to MeJA treatment. As shown in Figure 8, pretreatment with HT attenuated or completely blocked MeJA-induced aphid resistance, the activities of TI, CI, PPO, and PAL and the content of lignin, during plant response to aphid infestation. These results indicate the involvement of H₂S in MeJA-mediated defense response and aphid resistance.

FIGURE 7. Involvement of methyl jasmonate (MeJA) in melatonin-induced H2S accumulation in response to aphid resistance. In (a), the plant leaves were sprayed with 100 μM MeJA 12 h prior to aphid infestation. In (b), the plant leaves were sprayed with diethyldithiocarbamic acid (DIECA, 5 mM) 8 h prior to melatonin (100 μM) treatment. Twelve hours later, the leaves were infested with aphids. H2S contents were determined at indicated times. Values are the means of three replicates (±SDs). FW, fresh weight.

FIGURE 8. Involvement of H2S in methyl jasmonate (MeJA)-induced aphid resistance and defense response in watermelon leaves. The plant leaves were sprayed with hypotaurine (HT, 100 μM) 8 h prior to MeJA (100 μM) treatment. Twelve hours later, the plants were infested with aphids. On the 3rd and 7th day after the aphid infestation, the number of aphids was counted and the activities of defense-related enzymes and the content of lignin were measured on the 3rd and 7th day after the aphid infestation. Data of aphid number are expressed as means ± SDs (n = 15). Values of enzyme activities and lignin content are the means ± SD of three replicates. Means denoted with different letters differ significantly (p [less than] 0.05, Tukey's test). CI, chymotrypsin inhibitor; DW, dry weight; FW, fresh weight; PAL, phenylalanine ammonia lyase; PPO, polyphenol oxidase; TI, trypsin inhibitor.

To evaluate whether MeJA and H₂S induce melatonin biosynthesis and whether H₂S regulates MeJA biosynthesis in a feedback manner, we analyzed the response of melatonin to MeJA and NaHS and the response of MeJA to NaHS. Unexpectedly, MeJA did not induce significant changes in melatonin level within 3 days, but reduced melatonin level on the 7th day under both normal and aphid infestation conditions (Figure 9a). Under normal conditions, NaHS increased melatonin level within 3 days, but decreased melatonin level on the 7th day (Figure 9b). However, melatonin level was induced at 3 h and then was reduced by NaHS during aphid infestation response. Similarly, NaHS decreased the accumulation of MeJA under both normal and aphid infestation conditions (Figure 9c).

FIGURE 9. Effects of methyl jasmonate (MeJA) or NaHS on melatonin or MeJA accumulation in response to aphid infestation. (a) Kinetics of changes in melatonin content in response to MeJA and aphid infestation. The plant leaves were sprayed with 100 μM MeJA 12 h prior to aphid infestation. (b, c) Kinetics of changes in melatonin and MeJA content in response to NaHS and aphid infestation. The plant leaves were sprayed with 50 μM NaHS 12 h prior to aphid infestation. Values are the means ± SD of three replicates. FW, fresh weight.

DISCUSSION Melatonin induces watermelon resistance against aphid infestation

The latest research shows that exogenous melatonin application enhances aphid resistance of Nicotiana tabacum (Song et al., 2022). Consistent with this previous study, our data showed that exogenous application of melatonin-enhanced watermelon resistance against aphid infestation and this role of melatonin is dose dependent, whereas inhibition of melatonin biosynthesis by CPA decreased aphid resistance (Figure 1 and Figures S1 and S2). The protective effect of melatonin was attenuated with melatonin concentrations both higher and lower than the most effective concentration (around 100 μM). The increasing concern to minimize the use of pesticides and to switch onto sustainable and natural control strategies indicates the suitability of melatonin in combating aphids.

Higher plants have evolved a broad range of defensive mechanisms to reduce the threat of phytophagous insects. One common strategy used by plant species is the induction of activities of defense-related enzymes and biosynthesis of compounds that adversely affect herbivore performance (Gatehouse, 2002). Proteinase inhibitors, such as TI and CI, are rapidly induced in response to insect attacks, act on insect digestive enzymes once ingested into insect gut, and consequently inhibit insect growth and development (Chen et al., 2005). Polyphenol oxidases (PPOs) are antinutritive enzymes that oxidize phenolics to quinones, which decrease the nutritional quality of ingested plant tissues (Chen, 2008). Moreover, artificial feeding with PPO-treated dietary proteins can suppress insect growth (Felton et al., 1992). Accumulation of lignin can strengthen cell walls and therefore makes cell walls more difficult for leaf-eating herbivores to digest and piercing-sucking herbivores to penetrate (Mottiar et al., 2016; Naoumkina et al., 2010; Theis & Lerdau, 2003). In the current study, melatonin increased the activities of TI, CI, PPO, and PAL (an enzyme involved in lignin biosynthesis) and the accumulation of lignin during watermelon response to aphid infestation, suggesting that melatonin-induced aphid resistance is closely correlated with these defense-related enzymes and compounds (Figure 2).

MeJA is required for melatonin-induced aphid resistance of watermelon

Increasing evidence has demonstrated that the JA pathways induced by aphids during feeding play critical roles in regulating host-plant resistance against aphids (Yates-Stewart et al., 2020). Increased endogenous JA or its derivatives activate their receptors and subsequently induce the expression of numerous defense genes, leading to defense responses to aphid attack, such as the production of proteinase inhibitors (Morkunas et al., 2011). In line with the findings of previous studies (Ali & Agrawal, 2014; Qi et al., 2020; Selig et al., 2016), our data showed that pretreatment with MeJA-induced aphid resistance of watermelon in a dose-dependent manner and the most effective concentration is around 100 μM (Figure 4a,b), whereas inhibition of MeJA biosynthesis by DIECA decreased aphid resistance (Figures S1 and S2). Moreover, the induction of aphid resistance by MeJA is closely correlated with the increased activities of defense-related enzymes and accumulation of lignin (Figure 5a).

The cross talk between melatonin and MeJA in plant response to various abiotic and biotic stresses has been demonstrated. JA signaling pathway is involved in melatonin-induced Botrytis cinerea resistance of tomato fruit (Liu et al., 2019). In the current study, pretreatment with melatonin increased MeJA accumulation in watermelon plants in response to aphid feeding (Figure 3a), while application of MeJA failed to increase melatonin accumulation (Figure 9a). Moreover, inhibition of MeJA biosynthesis by DIECA pretreatment attenuated the melatonin-induced increase in defense-related enzyme activities and lignin content and subsequent aphid resistance (Figure 6). Taken together, these results indicate that MeJA is involved in melatonin-induced defense response and subsequent aphid resistance of watermelon plants.

MeJA-dependent H2S is involved in melatonin-induced aphid resistance

A growing body of evidence has shown that H₂S plays an important role in plant defense, particularly against fungal and bacterial pathogens (Choudhary et al., 2022; Vojtovi et al., 2021). As well as melatonin and MeJA, application of NaHS, the most widely used donor that directly and instantaneously releases H₂S in plants (Huang et al., 2021), also enhanced watermelon resistance against aphids in a dose-dependent manner and the most effective concentration is around 50 μM (Figure 4c,d), whereas inhibition of H₂S accumulation by HT decreased aphid resistance (Figures S1 and S2). To our knowledge, we provided the first evidence for the positive role of H₂S in regulating aphid resistance of plants. H₂S regulates plant defense response to microbial pathogens potentially by inducing the expression of defense-related genes and modulating regulating the activities of defense-related enzymes (Blackman & Eastop, 2007; Choudhary et al., 2022). Similarly, NaHS enhanced aphid resistance involves the induction of the activities of defense-related enzymes and the accumulation of lignin (Figure 5b).

Several recent studies have reported the cross talk between melatonin and H₂S in regulating plant response to abiotic stresses and H₂S may function downstream of melatonin. For instance, melatonin application enhances plant tolerance to heat, iron deficiency, and salinity, accompanied by increases in the accumulation of H₂S, while inhibition of endogenous H₂S accumulation by HT attenuated or abolished melatonin-induced this tolerance (Iqbal et al., 2021; Kaya et al., 2020). Consistent with the data of these studies, exogenous application of melatonin-induced increase in H₂S accumulation during watermelon response to aphid feeding (Figure 3b), while NaHS application failed to increase melatonin accumulation (Figure 9b). Furthermore, HT pretreatment attenuated the melatonin-induced increase in defense-related enzyme activities and lignin content and subsequent aphid resistance (Figure 6). Therefore, it is apparent that H₂S is involved in melatonin-induced aphid resistance of watermelon plants.

As two crucial signal molecules, the cross talk between JA and H₂S has been revealed in some physiological processes, such as stomatal movement, stomatal development, organ abscission, and ascorbate and glutathione metabolism (Deng et al., 2020; Hou et al., 2011; Li, Xiang & Wang, 2021; Shan et al., 2018). For instance, H₂S exerts its signaling role in the downstream of JA signaling to inhibit stomatal development and to promote stomatal closure in Arabidopsis (Deng et al., 2020; Hou et al., 2011). JA and H₂S also interact with each other to regulate plant defense against microbial pathogens (Choudhary et al., 2022). Here, exogenous MeJA induced H₂S accumulation in watermelon in response to aphid feeding (Figure 7a), while NaHS application failed to increase MeJA accumulation (Figure 9c). Furthermore, HT application attenuated the MeJA-induced increase in defense-related enzyme activities and lignin content and subsequent aphid resistance (Figure 8). These results suggest that H₂S acts as a downstream signaling molecule involved in MeJA-induced aphid resistance. In addition, pretreatment with DIECA prevented the induction of H₂S accumulation by melatonin (Figure 7b), suggesting that MeJA mediates melatonin-induced H₂S accumulation in response to aphid infestation.

CONCLUSIONS

To date, the signaling mechanisms underlying melatonin-induced aphid resistance of plants are still unclear. This study reveals an intricate signaling cascade of melatonin-induced aphid resistance of watermelon. Melatonin induces the accumulation of MeJA, which in turn increases H₂S production. H₂S further induces the activities of defense-related enzymes and the accumulation of lignin and subsequently enhances aphid resistance of watermelon. To the best of our knowledge, this is the first study of its kind to provide evidence for the positive role of H₂S in regulating aphid resistance and the involvement of MeJA-dependent H₂S signaling in melatonin-mediated aphid resistance of plants.

ACKNOWLEDGEMENTS

The authors thank the reviewers and editors for their great comments and feedback all along the review process.

FUNDING INFORMATION

The work was supported by the Earmarked Fund for China Agriculture Research System (CARS-25), the Young Science and Technology Star of Shaanxi (2022KJXX-80), the National Natural Science Foundation of China (31972479), the Science and Technology Innovation Team of Shaanxi (2021TD-32), the Science and Technology Plan Project of Xi'an (22NYYF027), and the Tang Scholar of Northwest A&F University (A1190021003).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflict of interest.

DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article.