H₂O₂ Functions as a Downstream Signal of IAA to Mediate H₂S-Induced Chilling Tolerance in Cucumber

Abstract

Hydrogen sulfide (H₂S) plays a crucial role in regulating chilling tolerance. However, the role of hydrogen peroxide (H₂O₂) and auxin in H₂S-induced signal transduction in the chilling stress response of plants was unclear. In this study, 1.0 mM exogenous H₂O₂ and 75 μM indole-3-acetic acid (IAA) significantly improved the chilling tolerance of cucumber seedlings, as demonstrated by the mild plant chilling injury symptoms, lower chilling injury index (CI), electrolyte leakage (EL), and malondialdehyde content (MDA) as well as higher levels of photosynthesis and cold-responsive genes under chilling stress. IAA-induced chilling tolerance was weakened by N, N′-dimethylthiourea (DMTU, a scavenger of H₂O₂), but the polar transport inhibitor of IAA (1-naphthylphthalamic acid, NPA) did not affect H₂O₂-induced mitigation of chilling stress. IAA significantly enhanced endogenous H₂O₂ synthesis, but H₂O₂ had minimal effects on endogenous IAA content in cucumber seedlings. In addition, the H₂O₂ scavenger DMTU, inhibitor of H₂O₂ synthesis (diphenyleneiodonium chloride, DPI), and IAA polar transport inhibitor NPA reduced H₂S-induced chilling tolerance. Sodium hydrosulfide (NaHS) increased H₂O₂ and IAA levels, flavin monooxygenase (FMO) activity, and respiratory burst oxidase homolog (RBOH1) and FMO-like protein (YUCCA2) mRNA levels in cucumber seedlings. DMTU, DPI, and NPA diminished NaHS-induced H₂O₂ production, but DMTU and DPI did not affect IAA levels induced by NaHS during chilling stress. Taken together, the present data indicate that H₂O₂ as a downstream signal of IAA mediates H₂S-induced chilling tolerance in cucumber seedlings.

1. Introduction

Cucumbers (Cucumis sativus L.) are typical light-loving and cold-sensitive plants, but they are mainly cultivated in solar greenhouses in northern China. When exposed to temperatures below 10 °C, cucumber plants generally suffer chilling injury (Ai et al.) [1]. Therefore, chilling is considered as a crucial limitation to growth and yield in cucumber production. Hydrogen sulfide (H₂S) is a novel gaseous signaling molecule that plays an important role in regulating plant growth and development and defense responses to various abiotic stresses. Previous studies revealed that H₂S upregulated the expression levels of mitogen-activated protein kinase (MAPK) and was involved in the upregulation of MAPK gene expression caused by cold stress [2]. The exogenous fumigation of H₂S or application of sodium hydrosulfide (NaHS, the H₂S donor), can relieve multiple abiotic stresses, such as chilling, heat, salinity, drought, hypoxia, and heavy metal toxicity [3]. We recently found that NaHS enhances the chilling tolerance of cucumber by scavenging reactive oxygen species (ROS), increasing CO₂ assimilation, and upregulating the expression of cold-responsive genes [4]. Some signaling molecules, such as nitrogen monoxide (NO), Ca²⁺, abscisic acid (ABA), and indol-3-acetic acid (IAA) are involved in H₂S-induced response to chilling stress in cucumber [4,5,6,7]. However, whether any other signaling molecules are involved in the process of H₂S-induced chilling tolerance, the relationship between these signaling molecules remains unclear.

Studies over the last decades have indicated that endogenous hydrogen peroxide (H₂O₂) is induced in plants after exposure to abiotic stress, such as low or high temperature, heavy metals, water stress, etc. [8,9,10]. H₂O₂ interacts with other plant growth regulators, such as auxins, gibberellins, cytokinins, etc. (as signaling molecules) synergistically or antagonistically, and it mediates plant growth and development and responses to abiotic stresses [10]. Pasternak et al. [11] suggested that the variation of PINOID gene expression triggered by H₂O₂ influenced the polar transport of auxin and might alter auxin homeostasis. The application of H₂O₂ induced the formation of adventitious roots in Linum usitatissimum by regulating endogenous auxin levels [12]. Zhu et al. [13] demonstrated that ethylene and H₂O₂ play an important role in triggering brassinosteroid-induced salt tolerance in tomato plants. Our recent study suggests that H₂O₂ is involved in H₂S-induced photoprotection in cucumber seedlings after exposure to chilling [14].

Auxin plays an essential role in the regulation of plant growth and development, but information about its role under chilling stress remains limited. Previous studies have revealed that changes in plant growth and development caused by cold stress are closely related to the intracellular auxin gradient, which is regulated by the polar deployment and intracellular trafficking of auxin transporters [15]. Recently, we found that IAA, a main auxin, could increase chilling tolerance by decreasing ROS accumulation, increasing the enzyme activities of photosynthesis and upregulating the expression of cold-responsive genes [4]. IAA also participates in the H₂S-mediated response to chilling stress in cucumber, and it controls the H₂O₂ in the growing part of the root [16]. Therefore, we speculate that crosstalk may exist among H₂O₂, IAA, and H₂S in response to chilling stress. To test this assumption, we investigated the effect of H₂O₂ and IAA on the ROS accumulation, photosynthesis, and relative expression of cold-responsive genes and the role of H₂O₂ and IAA in H₂S-induced chilling tolerance in cucumber seedlings.

2. Results

2.1. H₂O₂ Is Involved in H₂S-Induced Chilling Tolerance in Cucumber

To explore the effect of exogenous H₂O₂ on chilling tolerance in cucumber, we determined the maximum photochemical efficiency of PSII (F_v/F_m), actual photochemical efficiency of PSII (Φ_PSII), chilling injury index (CI), electrolyte leakage (EL), and photosynthetic rate (P_n) of cucumber seedlings, which were pretreated with different concentrations of H₂O₂ after exposure to 8/5 °C (day/night) for 24–72 h. As shown in Figure 1, H₂O₂ alleviated chilling injury symptoms in cucumber seedlings, and this alleviation effect was increased at low concentrations of H₂O₂ but was suppressed when the concentration exceeded 1.0 mM. The F_v/F_m, Φ_PSII, and P_n of H₂O₂-treated seedlings were much higher, and the CI and EL were much lower than 0 mM H₂O₂ (H₂O) treatments. These results reveal that H₂O₂ improves the chilling tolerance of cucumber seedlings, and its effect is concentration dependent. Thus, we use 1.0 mM H₂O₂ in further experiments.

We previously demonstrated that 1.0 mM NaHS markedly increased endogenous H₂O₂ accumulation, and H₂S-induced H₂O₂ plays an important role in CO₂ assimilation and photoprotection in cucumber [14,17]. Consistent with previous results, we found that NaHS induced endogenous H₂O₂ production. However, both N, N′-dimethylthiourea (DMTU, a H₂O₂ scavenger) and diphenyleneiodonium chloride (DPI, a H₂O₂ synthesis inhibitor) markedly inhibited the H₂S-induced increase in H₂O₂ biosynthesis and Respiratory burst oxidase homolog (RBOH1) mRNA abundance in seedlings under chilling stress (Figure 2a,b). NaHS obviously decreased the CI and EL and increased F_v/F_m and Φ_PSII, but the NaHS-induced decrease in CI and EL or increase in F_v/F_m and Φ_PSII in stressed seedlings were weakened by DMTU and DPI (Figure 2c–e). Therefore, we speculate that H₂O₂ is involved in the H₂S-induced response to chilling stress.

2.2. H₂O₂ Participates in IAA-Induced Chilling Tolerance in Cucumber

Our previous study demonstrated that IAA acts as a downstream signaling molecule and is involved in H₂S-induced chilling tolerance in cucumber seedlings [4]. To explore the interactions of H₂S, IAA, and H₂O₂ in response to chilling stress, we studied the interaction between H₂O₂ and IAA in the chilling stress response in cucumber. We first measured the EL, CI, and malondialdehyde (MDA) content in cucumber seedlings pretreated with 75 μM IAA, 1.0 mM H₂O₂, 5.0 mM DMTU + 75 μM IAA, 50 μM 1-naphthylphthalamic acid (NPA, a polar transport inhibitor of IAA) + 1.0 mM H₂O₂, or deionized water, after exposure to 8/5 °C for 48–72 h. Seedlings pretreated with IAA and H₂O₂ showed remarkably lower EL, CI, and MDA content than H₂O-pretreated seedlings during chilling stress (Figure 3a–c). The decrease in EL, CI, and MDA content in IAA treatment was blocked by DMTU, but the values in H₂O₂ pretreated seedlings were not significantly affected by the IAA polar transport inhibitor NPA. The IAA- and H₂O₂ pretreated seedlings exhibited distinctly less damage caused by chilling. The effects of IAA in mitigating in chilling damage in cucumber seedlings was weakened by DMTU, but NPA had minimal effect on H₂O₂-induced remission of chilling damage (Figure 3d).

Then, we detected the interactive effects of IAA and H₂O₂ on the mRNA levels of large and small subunits (rbcL, rbcS) of ribulose 1, 5-bisphosphate carboxylase/oxygenase (rubisco) and rubisco activase (RCA), as well as the P_n, F_v/F_m and Φ_PSII under chilling stress. Both IAA and H₂O₂ treatments revealed a marked increase in mRNA levels of rbcL, rbcS, and RCA (Figure 4a–c), and rbcL and RCA protein levels (Figure 4e), compared with H₂O treatment (p < 0.05). The application of DMTU distinctly repressed IAA-induced expression of rbcL, rbcS, and RCA, but NPA did not inhibit the effect of H₂O₂ on rbcL, rbcS, and RCA mRNA levels. Chilling stress significantly reduced the P_n of cucumber seedlings. After chilling treatment for 24 h, the decrease in P_n in cucumber seedlings was 77.6%, 53.9%, 58.6%, 74.2%, and 61.8% in the H₂O, IAA, H₂O₂, DMTU + IAA, and NPA+ H₂O₂ treatments respectively, compared to the control (Figure 4d). Figure 4f shows that F_v/F_m and Φ_PSII were markedly higher in IAA- and H₂O₂-treated than in H₂O-treated seedlings during chilling stress. The application of DMTU significantly weakened the IAA-induced increase in F_v/F_m and Φ_PSII, but NPA showed a minimal influence on the H₂O₂-induced variation of F_v/F_m and Φ_PSII. These data suggest that IAA and H₂O₂ mitigate the negative effect of chilling stress on the photosynthetic function by upregulating the mRNA and protein levels of the key photosynthetic enzymes and activating the photoprotective mechanism.

We also analyzed the effect of IAA and H₂O₂ on the relative expression of the cold responsive genes after seedlings were exposed to chilling stress for 24 h. IAA and H₂O₂ notably increased the mRNA levels of C-repeat-binding factor (CBF1), inducer of CBF expression (ICE1) and cold responsive (COR47) genes (Figure 5a–c) as well as CBF1 protein levels (Figure 5d) in cucumber seedlings under chilling stress. The increases in the mRNA and protein levels of the cold responsive genes in IAA-treated seedlings were dramatically weakened by DMTU, whereas those in H₂O₂-treated seedlings were minimally affected by NPA.

We found that 75 μM IAA remarkably increased the RBOH activity (Figure 6a) and H₂O₂ content (Figure 6b) in cucumber seedlings, and the increase was remarkable after treatment for 6 h. However, no remarkable differences in flavin monooxygenase (FMO) activity and IAA content were observed between H₂O₂- and H₂O-treated seedlings (Figure 7). At normal temperature, the H₂O₂-treated seedlings showed similar mRNA expressions of PIN1 and AUX2 to the H₂O-treated seedlings. After 9 h or 24 h of chilling stress, PIN1 and AUX2 mRNA levels markedly increased in both H₂O₂ and H₂O treatment, but the extent of the increase did not vary and showed no significant differences between H₂O₂ and H₂O_-treated seedlings (Supplemental Figure S1). All the above results indicate that IAA affects H₂O₂ signaling in cucumber seedlings under chilling stress. H₂O₂ might play a critical role in the IAA-induced positive response to chilling stress in cucumber seedlings.

2.3. Interaction of IAA and H₂O₂ in H₂S-Induced Chilling Tolerance in Cucumber

To further analyze the upstream and downstream relationship between IAA and H₂O₂ in H₂S-mediated plant stress response, we determined the effect of NPA on H₂S-induced H₂O₂ production and that of the H₂O₂ scavenger DMTU and H₂O₂ synthetic inhibitor DPI on H₂S-induced IAA biosynthesis. As shown in Figure 8, 1.0 mM NaHS markedly increased RBOH1 mRNA abundance and H₂O₂ accumulation. NPA significantly inhibited the increase in RBOH1 mRNA abundance and H₂O₂ content induced by NaHS, suggesting that IAA is involved in H₂S-induced H₂O₂ production. NaHS also upregulated FMO-like protein (YUCCA2) mRNA abundance, FMO activity, and IAA levels, but DMTU and DPI had minimal effects on H₂S-induced IAA biosynthesis in cucumber leaves (Figure 9). Combining the results of Figure 2, it is further inferred that H₂O₂, a downstream component of IAA, is involved in H₂S-induced chilling tolerance in cucumber seedlings.

3. Discussion

Chilling is a major abiotic stress that affects the growth, development, and geographical distribution of plants [18,19]. Low-temperature stress mainly affects light energy utilization and photosynthetic efficiency by destroying electron transport chains in chloroplasts and mitochondria, leading to ROS accumulation, and eventually inducing cell membrane damage in plants [20]. H₂S, as a major gaseous transmitter, plays a critical role in plant resistance to various stress conditions, such as low temperature, salt, drought, and heavy metals [21,22]. The application of exogenous H₂S can enhance chilling tolerance in Arabidopsis thaliana [1], hawthorns [23], and cucumbers [3]. ABA, NO, Ca²⁺, and SA are involved in H₂S-induced resistance to abiotic stresses in plants [24,25,26]. Recently, we verified that H₂S interacts with NO, ABA, Ca²⁺, IAA, and SA to enhance the chilling tolerance in cucumbers [4,5,6,7,27]. However, whether H₂O₂ and IAA exhibit synergistic effects on the H₂S-mediated plant stress response remains unclear.

Previous studies have revealed that H₂O₂ is a key molecule of signal transduction and regulates various physiological metabolic processes. For example, H₂O₂ recruited the promoter of the senescence-related transcription factor WRKY53, which in turn activated WRKY53 transcription and led to a senescence of Arabidopsis [28]. The H₂O₂ response gene (HRG1/2) could quickly respond to exogenous or endogenous H₂O₂ and further regulated Arabidopsis seed germination [29]. Islam et al. proved that by inducing production of the reactive carbonyl species (RCS), H₂O₂ could induce stomatal closure of guard cells in Arabidopsis [30]. Moreover, H₂O₂ responds to many abiotic stress of plants. Sun et al. showed that as a signal, on the one hand, Respiratory burst oxidase homologue-dependent H₂O₂ (RBOH-H₂O₂) enhanced the heat tolerance of heat sensitive tomato. On the other hand, RBOH-H₂O₂ regulated the activities of antioxidant enzymes to control the total H₂O₂ at a level conducive to heat stress memory, which in turn maintained a lower level of total H₂O₂ during the future heat stress challenge [8]. H₂O₂ also could interact synergistically with other hormones or regulators, such as IAA, ABA, SA, MeJA, etc., mediating the response to abiotic stress in plants [10,31]. Recently, our results showed that H₂O₂ induced CO₂ assimilation and photoprotection in cucumber seedlings during chilling stress [14]. In this study, we found that H₂O₂ increased the chilling tolerance of cucumber seedlings (Figure 1), suggesting that the response of H₂O₂ to chilling stress is consistent with previous studies. NaHS significantly enhanced H₂O₂ levels, RBOH1 mRNA abundance, and chilling tolerance in cucumber seedlings. The H₂O₂ scavenger DMTU and inhibitor of H₂O₂ synthesis DPI decreased H₂S-induced H₂O₂ accumulation and chilling tolerance (Figure 2). These results indicate that H₂O₂ may crosstalk with H₂S to improve the chilling stress response in cucumber seedlings.

Auxin is a major phytohormone that controls various aspects of plant growth and development, including cell division and elongation, tissue patterning, and the response to environmental stimuli [32,33], but knowledge about its role and interaction with other signals under chilling stress is limited. Previous investigations have indicated that chilling-induced variation in plant growth and development is closely related to the intracellular auxin gradient. Chilling stress promotes auxin biosynthesis or changes auxin gradient distribution, thus affecting the root gravity response in Arabidopsis, rice, and poplar [15,34,35,36]. Recently, we learned that NaHS increased endogenous IAA accumulation and improved chilling tolerance. The IAA polar transport inhibitor NPA suppressed H₂S-induced chilling tolerance. IAA reduced the negative effects of chilling stress on growth and photosynthesis, but it showed minimal effects on endogenous H₂S levels. H₂S scavengers did not influence the chilling tolerance induced by IAA [4]. Here, we observed that IAA-induced chilling tolerance was repressed by the H₂O₂ scavenger DMTU, but the IAA inhibitor NPA did not affect H₂O₂–induced tolerance to chilling stress (Figure 3, Figure 4 and Figure 5). IAA significantly enhanced endogenous H₂O₂ synthesis, but H₂O₂ showed minimal effects on endogenous IAA level in cucumber seedlings (Figure 6 and Figure 7). Thus, we speculate that IAA depends on the H₂O₂ signaling pathway in the regulation to chilling stress response. In addition, NPA significantly decreased H₂S-induced RBOH1 and H₂O₂ levels (Figure 8), whereas DMTU and DPI showed no marked effect on H₂S-induced YUCCA2 mRNA abundance, FMO activity, or IAA levels (Figure 9). These results suggest that H₂O₂ lies downstream of IAA in the regulation of H₂S to the chilling stress response.

Based on previous studies and the above results, we proposed a model of H₂O₂ and IAA regulating the H₂S-mediated chilling stress response in cucumber seedlings. Figure 10 shows that endogenous H₂S induced by chilling stress or the application of exogenous NaHS, H₂O₂, and IAA all enhanced chilling tolerance in cucumber seedlings by scavenging excessive ROS, improving photosynthetic capacity, and upregulating the mRNA and protein levels of cold responsive genes. Chilling stress-induced or exogenous H₂S promotes IAA generation, and IAA further triggers H₂O₂ accumulation and subsequently increases chilling tolerance. Thus, H₂O₂ may act as a downstream signal of IAA and play a significant role in H₂S-mediated chilling stress tolerance in cucumber seedlings. Further studies using advanced molecular techniques and mutants are required to better reveal the mechanisms and interactions of H₂S-, IAA-, and H₂O₂-induced chilling tolerance in plants.

In summary, H₂O₂ and IAA markedly improved the chilling tolerance of cucumber seedlings, as illustrated by the decrease in stress-induced CI and EL, the increase in CO₂ assimilation, and the upregulation in the level of cold-responsive genes. Even more importantly, our results first confirmed that H₂O₂ interacts with IAA signaling and is jointly involved in H₂S-induced chilling tolerance in cucumber. Moreover, H₂O₂ may act as a downstream signaling molecule of IAA, and it plays a critical role in H₂S-mediated chilling stress response in cucumber.

4. Materials and Methods

4.1. Plant Materials and Growth Condition

“Jinyou 35” cucumber (Cucumis sativus L.) seedlings were used in the current study. After soaking and germinating, the seeds were sown in nutrition bowls filled with seedling substrate, which consisted of peat, vermiculite, and perlite (5:3:1, v/v), and then transferred to a climate chamber with a photon flux density (PFD) of 600 μmol m⁻²·s⁻¹, a 26/17 °C thermoperiod, an 11 h photoperiod, and 80% relative humidity.

4.2. Experimental Design

4.2.1. Effect of H₂O₂ on the Chilling Tolerance of Cucumber Seedlings

The seedlings with two leaves were foliar sprayed with 0 (control), 0.01, 0.1, 1.0, 2.0, and 3.0 mM H₂O₂, respectively. Twenty-four hours later, the pretreated seedlings were exposed to 8/5 °C to analyze the CI, EL, P_n, F_v/F_m, and Φ_PSII.

4.2.2. Effect of H₂O₂ Scavenger or Inhibitor on H₂S-Induced H₂O₂ Biosynthesis and Chilling Tolerance

The seedlings were pretreated with 1.0 mM NaHS, (a H₂S donor), 5.0 mM DMTU (a H₂O₂ scavenger), 100 μM DPI (a H₂O₂ synthesis inhibitor), 5.0 mM DMTU + 1.0 mM NaHS, 100 μM DPI + 1.0 mM NaHS, or deionized water (H₂O). Twenty-four hours later, the pretreated seedlings were subjected to 8/5 °C for 9–72 h to assay the biosynthesis of H₂O₂, EL, CI, F_v/F_m, Φ_PSII and relative expression of cold-responsive genes. The H₂O treatment at normal temperature served as the control.

4.2.3. Interaction between IAA and H₂O₂ in Response to Chilling Stress

The seedlings were pretreated with 75 μM IAA, 1.0 mM H₂O₂, 5.0 mM DMTU + 75 μM IAA, 50 μM NPA (a polar transport inhibitor of IAA) +1.0 mM H₂O₂, or deionized water (H₂O). At 24 h after pretreatment, the seedlings were exposed to 8/5 °C to assay the P_n, fluorescence parameters, gene expression and protein expression of key photosynthesis enzymes, and relative expression of cold-responsive genes. H₂O treatment under normal conditions served as the control.

4.2.4. Effect of IAA Inhibitor on H₂S-Induced H₂O₂ Biosynthesis in Cucumber Seedlings

The seedlings were pretreated with 1.0 mM NaHS, 50 μM NPA, 50 μM NPA + 1.0 mM NaHS, or deionized water (H₂O). Twenty-four hours later, the pretreated seedlings were subjected to 5 °C for 9 h to assay the relative expression of RBOH1 and H₂O₂ content.

4.2.5. Effect of Scavengers and Synthetic Inhibitors of H₂O₂ on H₂S-Induced IAA Biosynthesis in Cucumber Seedlings

The seedlings were pretreated with 1.0 mM NaHS, 5.0 mM DMTU, 100 μM DPI, 5.0 mM DMTU + 1.0 mM NaHS, 100 μM DPI + 1.0 mM NaHS, or deionized water (H₂O). Twenty-four hours later, the pretreated seedlings were subjected to 5 °C for 9 h to assay the FMO activity, YUCCA2 mRNA abundance, and IAA content.

4.3. CI, EL, and MDA Measurements

The chilling stressed cucumber seedlings were graded based on the Semeniuk et al. [37] standard, and the CI was calculated according to the following formulas: CI = Σ (plants of different grade × grade)/[total plants × 5 (the maximum grade)].

EL was measured as described by Dong et al. [38]. Leaf discs (0.2 g) were incubated at 25 °C in 20 mL deionized water for 3 h, and the electrical conductivity (EC1) was estimated using a conductivity meter (DDB-303A, Shanghai, China). Afterwards, the leaf discs were boiled for 30 min and then cooled to detect EC2. EL was calculated according to the following formula: EL= EC1/EC2 × 100.

MDA content was determined using the thiobarbituric acid (TBA) colorimetric method as described by Heath and Packer [39].

4.4. Detection of P_n and Chlorophyll Fluorescence

The P_n was determined using a portable photosynthetic system (Ciras-3, PP-systems International, Hitchin, Hertfordshire, UK). Constant PFD (600 μmol·m⁻²·s⁻¹), CO₂ concentration (380 mg·L⁻¹), and leaf temperature (25 ± 1 °C) were maintained during the assessment. F_v/F_m was measured after seedlings were dark-adapted for 45 min, and the Φ_PSII was determined after leaves were light-adapted for 30 min using a portable pulse-modulated fluorometer (FMS-2, Hansatech, King’s Lynn, Norfolk, UK). The chlorophyll fluorescence parameters were calculated according to Demmig-Adams and Adams [40] and Maxwell [41] as follows: F_v/F_m = (F_m−F₀)/F_m′; Φ_PSII = (F_m′−F_s)/F_m′. Chlorophyll fluorescence imaging was visualized using a chlorophyll fluorescence imaging system (Imaging PAM, Walz, Wurzburg, Germany) with a computer-operated PAM-control system [42].

4.5. Detection of H₂O₂ Content and RBOH Activity

H₂O₂ content was determined using an H₂O₂ kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). RBOH activity was detected with an ELISA kit (Jiangsu Meimian Industrial Co. Ltd., Yancheng, China) according to the instructions.

4.6. IAA Content and FMO Activity Assay

IAA content was determined by the method of Zhang et al. [4]. In brief, 0.3 g sample ground with liquid nitrogen and extracted thrice with 80% methanol (containing 30 μg·mL⁻¹ sodium diethyldithiocarbamate). Samples were centrifuged (7155 g, 10 min, 4 °C) to obtain the supernatant by rotary evaporation (Shanghai EYELA, N-1210B, Shanghai, China) at 38 °C. The residue was washed with 5 ml of PBS (pH = 8, 0.2 M) and 4 mL of trichloromethane, shaken for 20 min, and allowed to stand for 30 min to remove pigment present in the trichloromethane. The resulting residues were added to 0.15 g of polyvinylpolypyrrolidone (PVPP) to remove phenols. Then, the samples were centrifuged at 7155 g for 10 min, and the resulting supernatant was re-extracted with ethyl acetate thrice and dried with a rotary evaporator in vacuo at 36 °C. The dried material was dissolved in 1.0 mL mobile phases (methanol: 0.04% acetic acid = 45:55, v/v), and the filtrate was used for HPLC–MS (Thermo Fisher Scientific, TSQ Quantum Access, San Jose, CA, USA) analysis followed by the method of Zhang et al. [4].

Flavin monooxygenase (FMO) activity was estimated using an ELISA kit (Jiangsu Meimian Industrial Co. Ltd., Yancheng, China). In brief, the FMO1 antibody was conjugated with standard, sample, and horseradish peroxidase (HRP)-labeled detection antibody and incubated, aspirated, and washed. Then, chromogen solution was added, and the reaction was terminated with sulfuric acid solution. The absorbance was detected at 450 nm with a microplate reader, and FMO activity was calculated using the standard curve [4].

4.7. Quantitative Real-Time PCR Analysis

Total RNA was extracted from cucumber leaves using an RNA extraction kit (TRIzol; Tiangen, Beijing, China). The isolated RNA was reverse transcribed with the PrimeScript^® RT Master Mix Perfect Real Time (TaKaRa, Dalian, China). qRT-PCR was performed using the TransStart^® TipTop Green q-PCR SuperMix (Cwbio, Beijing, China). The relative expression levels were standardized to those of cucumber β-actin gene (Solyc11g005330). The qRT-PCR primers are shown in Supplemental Table S1.

4.8. SDS-PAGE and Immunoblot Analysis

The extracted total protein of samples was separated using a 10% SDS-PAGE gel, and the resulting proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membrane was blocked for 2 h with 5% (w/w) skimmed milk and then incubated with the primary antibody followed by a horseradish peroxidase-conjugated anti-rabbit IgG antibody (ComWin Biotech Co., Ltd., Beijing, China) for 2 h. Finally, the immunoreaction was tested using the eECL Western blot Kit (CW00495, ComWin Biotech Co., Ltd., Beijing, China) and the ChemiDoc™ XRS imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Primary antibodies against RbcL and RCA (ATCG00490, AT2G39730) were obtained from PhytoAB Co. Ltd. (San Francisco, CA, USA), and the CBF1 antibody was obtained from GenScript Co., Ltd. (Nanjing, China).

4.9. Statistical Analysis

The whole experiment was performed in triplicate, and the results shown are the mean ± standard deviation (SD). Statistical analysis was performed with DPS software, and the comparison of treatments was based on the analysis of variance by Duncan’s multiple range test (DMRT) at a significance level of 5% (p < 0.05).