Hydrogen Sulfide Improves the Cold Stress Resistance through the CsARF5-CsDREB3 Module in Cucumber

As an important gas signaling molecule, hydrogen sulfide (H2S) plays a crucial role in regulating cold tolerance. H2S cooperates with phytohormones such as abscisic acid, ethylene, and salicylic acid to regulate the plant stress response. However, the synergistic regulation of H2S and auxin in the plant response to cold stress has not been reported. This study showed that sodium hydrosulfide (NaHS, an H2S donor) treatment enhanced the cold stress tolerance of cucumber seedlings and increased the level of auxin. CsARF5, a cucumber auxin response factor (ARF) gene, was isolated, and its role in regulating H2S-mediated cold stress tolerance was described. Transgenic cucumber leaves overexpressing CsARF5 were obtained. Physiological analysis indicated that overexpression of CsARF5 enhanced the cold stress tolerance of cucumber and the regulation of the cold stress response by CsARF5 depends on H2S. In addition, molecular assays showed that CsARF5 modulated cold stress response by directly activating the expression of the dehydration-responsive element-binding (DREB)/C-repeat binding factor (CBF) gene CsDREB3, which was identified as a positive regulator of cold stress. Taken together, the above results suggest that CsARF5 plays an important role in H2S-mediated cold stress in cucumber. These results shed light on the molecular mechanism by which H2S regulates cold stress response by mediating auxin signaling; this will provide insights for further studies on the molecular mechanism by which H2S regulates cold stress. The aim of this study was to explore the molecular mechanism of H2S regulating cold tolerance of cucumber seedlings and provide a theoretical basis for the further study of cucumber cultivation and environmental adaptability technology in winter.


Introduction
Cucumber (Cucumis sativus L.) is one of the most important economic crops worldwide. The cultivation and yield of cucumbers in China have ranked among the top in the world for many years. Cucumbers are typical cold-sensitive plants and are generally grown in solar greenhouses in northern China. Because of the extreme low-temperature conditions, cucumbers in greenhouses are prone to cold injury in winter and early spring. Cucumbers with cold injury showed inhibited growth, wilted and died in severe cases. Therefore, it is of great practical significance to study the effects of low-temperature stress on cucumber growth and development and the response mechanism of cucumber to low-temperature stress.
Hydrogen sulfide (H 2 S) is a gaseous compound recognized as the third gas signaling molecule discovered after nitric oxide and carbon monoxide [1,2]. H 2 S has been found to be widespread in mammals and has important cellular protective effects [3,4]. However, H 2 S is also highly toxic. Low concentrations of H 2 S can affect the eyes, respiratory system and central nervous system. Inhaling hydrogen sulfide in small concentrations can be fatal [5,6]. In agricultural production, the application of an appropriate concentration of H 2 S can regulate plant growth and development, such as germination, maturation, root development, senescence and defense [7][8][9]. Numerous investigations have determined that H 2 S plays a key role in the regulation of abiotic stress responses, including cold tolerance [8,[10][11][12]. Low temperatures trigger H 2 S biosynthesis [13,14]. H 2 S is found to alleviate cold stress tolerance in many plant species, although the mechanisms remain elusive [11,[13][14][15]. Several reports show that H 2 S modulates cold stress response, possibly through mitogen-activated protein kinase (MAPK) signaling [11,16,17]. In addition to H 2 S, phytohormones, especially auxin, also play a vital positive regulatory role in cold stress response [18,19].
Auxin is involved in plant growth and development in various aspects, including cell division and elongation, tissue patterning and the response to environmental stimuli [20,21]. Since auxin was identified for the first time in the 1930s as indole-3-acetic acid, there has been a major breakthrough in the molecular mechanisms of auxin perception and signal transduction. Many genetic and biochemical approaches have elucidated that the TRANSPORT INHIBTOR RESPONSE1 (TIR1) protein functions as the receptor to perceive auxin signaling based on the reduced auxin response of tir1 mutations [22,23]. Additionally, the SCF TIR1 ubiquitin-ligase complex is regarded as a central regulator of auxin signaling, and it-mediated proteolysis of auxin/indole acetic acid (Aux/IAA) proteins is responsible for auxin signaling transduction [24,25]. In this signaling pathway, Aux/IAA proteins are direct targets of TIR1. Aux/IAA proteins directly interact with auxin response factors (ARFs) to repress their activities [26]. Upon exposure to auxin, the F-box protein TIR1 recruits the Aux/IAA proteins for degradation, which leads to the release of various auxin response factors, including Small Auxin-up RNAs (SAURs), GH3s and Aux/IAAs, and consequently regulates diverse auxin-mediated plant growth [27,28].
In this study, the molecular mechanisms by which H 2 S regulates cold stress response in cucumber were explored. The study suggested that H 2 S treatment could improve cold resistance and auxin content of cucumber. CsARF5, a transcriptional regulator in auxin signaling, was responsive to cold stress and H 2 S treatments, and overexpression of CsARF5 improved the cold stress tolerance of cucumber. Further studies indicated that CsARF5 modulated cold stress response by directly activating the expression of the dehydrationresponsive element-binding (DREB)/C-repeat binding factor (CBF) gene CsDREB3.

Sodium Hydrosulfide (NaHS) Improves Cold Tolerance in Cucumber Seedlings
A previous study demonstrated that NaHS could improve the cold tolerance of cucumber seedlings in a concentration-dependent manner, and 1.0 mM NaHS treatment showed a very significant difference compared with the control [60]. Here, Figure 1 shows that 1.0 mM NaHS significantly reduced cold stress injury, accumulation of H 2 O 2 and superoxide anion (O 2 .− ), as well as electrolyte leakage (EL) in cucumber seedlings after exposure to 5 • C for 48 h. However, 0.15 mM H 2 S scavenger hypotaurine (HT) increased cold stress injury, H 2 O 2 , O 2 .− and EL, compared with the deionized water (H 2 O, as a comparison)-treated seedlings ( Figure 1A-F). The mRNA abundance of CsCBF1 and CsCOR in NaHS-treated seedlings also increased by 1.38-fold and 3.14-fold, respectively, under cold stress, but no obvious differences were observed between H 2 O and HT treatments ( Figure 1G,H). Therefore, the results further confirmed that H 2 S improves cold tolerance in cucumber.

Figure 1.
Effects of NaHS and HT on the cold tolerance of cucumber seedlings. Cucumber seedlings were treated with NaHS, HT or deionized water for 24 h and subsequently were exposed to cold stress. (A) Phenotypic characterization of cucumber seedlings before (control) and after cold stress (5 • C for 48 h). Each treatment contained 5-10 cucumber seedlings. and CsCOR genes in cucumber seedlings under cold stress treatment for 6 h. qRT-PCR was performed simultaneously with three biological replicates and three technical replicates. The value of the water treatment was used as the reference and was set to 1. Error bars denote standard deviations. Different letters indicate significant differences (p < 0.05) based on Duncan's multiple range tests.

NaHS Treatment Affects Auxin Signaling
To explore the molecular mechanism by which H 2 S improves cold resistance in cucumber, transcriptome analyses of cucumber seedlings treated with NaHS and H 2 O were performed. A total of 1952 cucumber genes were analyzed from transcriptome data ( Figure 2A). Among these cucumber genes, 118 genes were downregulated, and 54 genes were upregulated ( Figure 2A). The upregulated genes were further analyzed ( Figure 2B). One of the genes that caught our attention was an auxin response gene (accession number: CsaV3_3G045690, Figure 2B). The NCBI database comparison found that it was the CsARF5 gene. To study the effect of NaHS on auxin signaling, the change in auxin content in cucumber seedlings treated with NaHS or HT was estimated. NaHS (1.0 mM) markedly increased endogenous indole-3-acetic acid (IAA) accumulation and flavin monooxygenase (FMO, a key enzyme in auxin synthesis) activity. However, HT treatment revealed lower or similar IAA content and FMO activity compared with H 2 O treatment ( Figure 3A,B). In addition, NaHS treatment also significantly upregulated the mRNA levels of CsARF5 and CsDREB3, while HT-treated seedlings showed no marked differences in the relative mRNA expression of CsARF5 and CsDREB3 relative to the H 2 O treatment ( Figure 3C,D). These data indicate that H 2 S affects auxin signaling in cucumber seedlings under cold stress. Expression of the CsDREB3 gene in cucumber seedlings treated with NaHS or HT under cold stress treatment for 6 h. qRT-PCR was performed simultaneously with three biological replicates and three technical replicates. The value of 0 h was used as the reference and was set to 1. Error bars denote standard deviations. Different letters indicate significant differences (p < 0.05) based on Duncan's multiple range tests.

IAA Treatment Improves Cold Resistance of Cucumber Seedlings
A previous study showed that 75 µM IAA enhances the cold tolerance of cucumber seedlings [60]. Here, Figure 4 shows that 75 µM IAA significantly reduced the EL, and accumulation of H 2 O 2 and O 2 .− caused by cold stress, while 50 µM 1-naphthylphthalamic acid (NPA, a polar transport inhibitor) treatment showed no remarkable difference relative to H 2 O treatment under cold stress ( Figure 4A-F). As an auxin response factor, the relative mRNA expression of CsARF5 was upregulated in IAA-treated seedlings under cold stress. However, no remarkable difference was found in the mRNA expression of CsARF5 between the NPA and H 2 O treatments ( Figure 4G). The mRNA expression levels of CsCBF1 and CsCOR were significantly upregulated in IAA-treated seedlings but downregulated or not influenced in HT-treated seedlings compared with H 2 O-treated seedlings when exposed to cold stress ( Figure 4H,I). The latest results are in keeping with the earlier findings [60], so the results further confirm that IAA enhances cold tolerance in cucumber.

CsARF5 Positively Regulates Cold Stress Tolerance of Cucumber
qRT-PCR results showed that cold stress increased the mRNA abundance of the CsARF5 gene, and the expression reached a peak after seedlings were exposed to cold for 3 h and then decreased gradually ( Figure 5A). Compared with the control and HT treatments, NaHS treatment further increased the expression of CsARF5 ( Figure 5B). These results demonstrate that CsARF5 is responsive to cold stress and H 2 S treatment.  To further explore the role of CsARF5 in response to cold stress in cucumber, we obtained CsARF5 transgenic cucumber leaves through Agrobacterium-mediated transient genetic transformation (Supplemental Figure S1A). Then, the accumulation of reactive oxygen species (ROS) in transgenic leaves after exposure to cold stress for 12 h were observed, using nitroblue tetrazolium (NBT) and 3, 3-diaminobenzidine (DAB) staining. The results showed that the accumulation of ROS in cucumber leaves overexpressing CsARF5 (CsARF5) was lower than that of the empty vector control (WT) under cold stress ( Figure 5C,D). In addition, the contents of O 2 ·− and H 2 O 2 were measured with biochemical analysis, and the results were in agreement with the NBT and DAB staining images ( Figure 5E,F). qRT-PCR results revealed that CsARF5 overexpression upregulated the mRNA level of cold stress-responsive genes CsDREB3, CsCBF1 and CsCOR ( Figure 5G-I). These data suggest that CsARF5 is a positive regulator of cold stress response.

HT Treatment Affects CsARF5-Mediated Cold Stress Tolerance
Considering that H 2 S induces the expression of CsARF5, and that CsARF5 positively regulates cold stress resistance, the role of CsARF5 in H 2 S-mediated cold stress was further explored. The H 2 S scavenger HT was applied to CsARF5-overexpressing cucumber leaves to observe ROS accumulation. The NBT and DAB staining results showed that the application of HT alleviated the CsARF5-decreased ROS accumulation in detached cucumber leaves ( Figure 6A,B). The biochemical analysis for O 2 .− and H 2 O 2 was consistent with the NBT and DAB staining results ( Figure 6C,D). qRT-PCR results showed that the application of HT inhibited the promotion of CsDREB3, CsCBF1 and CsCOR mRNA expression levels caused by CsARF5 ( Figure 6E-G). These results suggest that the regulation of the cold stress response by CsARF5 depends on H 2 S.

Overexpression of CsDREB3 Enhances Cold Stress Tolerance of Cucumber
DREB/CBF TFs play essential roles in the regulation of the plant cold stress response [61,62]. CsDREB3 was induced by NaHS ( Figure 2B), which prompted us to explore whether CsDREB3 was involved in the cold stress response in cucumber. As shown in Figure 7A, cold stress induced the expression of the CsDREB3 gene, and the expression reached a peak at 3 h, and then decreased gradually. Compared with the control and HT treatments, NaHS treatment further increased the expression of CsDREB3 ( Figure 7B).
To investigate the role of CsDREB3 in response to cold stress in cucumber, CsDREB3 transient transgenic cucumber leaves were obtained (Supplemental Figure S1B). NBT and DAB staining results showed that overexpression of CsDREB3 decreased ROS accumulation after cold stress treatment ( Figure 7C,D). In addition, O 2 .− and H 2 O 2 detection results also revealed that the accumulation of O 2 .− and H 2 O 2 was significantly lower in leaves of overexpressing CsDREB3 than in WT leaves ( Figure 7E,F). qRT-PCR results suggested that overexpression of CsDREB3 increased the expression of CsCBF1 and CsCOR ( Figure 7G,H). These data demonstrate that CsDREB3 positively regulates the cold tolerance of cucumber.

CsARF5 Directly Activates the Expression of CsDREB3
CsARF5 acts as an auxin response factor and can bind to the AuxRE motif in the promoters of target genes [27]. Considering the similar expression patterns of CsARF5 and CsDREB3 in cold stress and H 2 S treatment, as well as the key role of DREB/CBF TFs in the regulation of the cold stress response, we hypothesized that CsARF5 might be involved in the cold stress response by mediating the expression of CsDREB3. Then, the sequence of the CsDREB3 gene promoter region was analyzed, and a putative AuxRE motif was found ( Figure 8A). Fortunately, the direct binding between the CsARF5 protein and the promoter of CsDREB3 was detected by electromobility shift assay (EMSA) ( Figure 8B). To test how CsARF5 regulated the expression of CsDREB3, dual luciferase assays in tobacco leaves were performed. The CsARF5 effector construct was expressed under the 35S promoter, and the promoter of CsDREB3 was fused to the Luc gene as a reporter ( Figure 8C). The results showed that co-expression of 35Spro:CsARF5 with CsDREB3pro: Luc led to an obvious increase in luminescence intensity ( Figure 8D,E), while the binding site was mutated, and the activation was abolished ( Figure 8D,E). These results suggest that CsARF5 transactivates the expression of CsDREB3 in cucumber.

Discussion
H 2 S, as a gaseous signaling molecule, plays a crucial role in plant relevance to various stress conditions, such as low temperature, salt, drought and heavy metals [12,63]. A report showed that the exogenous application of the H 2 S donor NaHS could effectively improve plant growth and stress response [64]. In this study, NaHS treatment reduced the accumulation of ROS and activated the expression of cold stress-responsive genes, thus improving the cold-tolerance of cucumber seedlings (Figure 1), suggesting that the response of H 2 S to cold stress is consistent in different species [64]. Previous reports indicate that H 2 S and phytohormones have synergistic effects on the H 2 S-mediated plant stress response [65]. For example, H 2 S interacts with abscisic acid (ABA) and ethylene and is involved in the plant response to drought stress and in the regulation of stomatal closure [66,67]. Salicylic acid (SA) may play a key role in H 2 S-alleviated heavy metal stress and low-temperature stress [68,69]. Jasmonic acid (JA) stimulation increases the H 2 S level of protective cells and induces stomatal closure in Vicia faba [70]. Here, the study found that NaHS treatment promoted IAA synthesis and upregulated the expression of auxin-responsive genes (Figures 2 and 3), indicating that H 2 S may crosstalk with auxin to regulate the cold stress response of cucumber.
Several plant hormones, such as JA, SA and ethylene, have been shown to play key roles in the plant's response to cold stress [66][67][68][69][71][72][73]. However, the role of auxin under cold stress is limited. Auxin is a crucial phytohormone that is involved in a variety of plant physiological and developmental processes, including the regulation of the cold stress response [74,75]. Previous investigations have determined that cold stress promotes auxin biosynthesis or changes the auxin gradient distribution, thus affecting the root gravity response in Arabidopsis, rice and poplar [18,19,76,77]. A recent study showed that the auxin signaling repressor IAA14 plays an important role in integrating microRNAs with auxin and cold reactions in Arabidopsis [78]. However, studies on the effects of auxin on plant cold stress response are not sufficient. This study showed that the application of auxin improved cold resistance, whereas the application of the polar transport inhibitor NPA slightly reduced the cold resistance of cucumber seedlings (Figure 4), demonstrating that auxin is a positive regulator of the cold stress response.
As a master regulator of auxin signaling, ARF TFs have been functionally characterized in Arabidopsis, rice and populus trichocarpa [27,31,32,79]. An increasing number of studies have indicated that ARFs regulate multiple plant developmental processes such as root growth [40,41], flower development [46,47], senescence [48] and stress response [19,80]. Among these processes, the auxin-regulated cold stress response has become a major focus of biotechnology in cucumber, with the task of identifying the cold resistance genes and improving the yield of cucumber. In this study, an ARF TF, CsARF5, was isolated from cucumber, and its expression was induced by NaHS and cold stress treatments, suggesting that CsARF5 may be involved in H 2 S-mediated cold tolerance ( Figure 5A, B).
To investigate the functions of CsARF5, transgenic cucumber leaves overexpressing CsARF5 were generated (Supplemental Figure S1A). As hypothesized, the physiology and genetic analysis indicated that the overexpression of CsARF5 decreased the accumulation of ROS and increased the expression level of cold stress-responsive genes, thus improving the cold stress resistance of cucumber ( Figure 5). These results suggest that CsARF5 positively regulates the cold stress tolerance of cucumber. Meanwhile, the H 2 S scavenger HT was applied to CsARF5-overexpressing cucumber leaves to study the role of CsARF5 in H 2 S-mediated cold stress. The results showed that HT inhibited the cold stress resistance increased by CsARF5 (Figure 6), indicating that the regulation of the cold stress response by CsARF5 depends on H 2 S. DREB/CBF proteins play important roles in the regulation of the plant cold stress response [64,65]. In Arabidopsis, DREB1A, DREB2C, CBF1, CBF2 and CBF3 are involved in the cold stress response [62,[81][82][83]. In rice, OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1F, OsDREB2B and OsDREBL are responsive to cold stress treatment [84][85][86][87]. Here, cucumber DREB genes, namely CsDREB3, were induced by NaHS and cold stress treatments, and overexpression of CsDREB3 significantly enhanced the cold stress tolerance of cucumber (Figure 7, Supplemental Figure S1B), revealing that CsDREB3 is a positive regulator of cold stress.
ARF5 is known to function as a transcriptional activator [27]. The remarkable expression of CsDREB3 in CsARF5 transgenic leaves prompted us to consider whether CsARF5 can directly regulate CsDREB3 expression ( Figure 5G). The putative AuxRE elements that were recognized by CsARF5 were searched in the promoter region of CsDREB3. Fortunately, one AuxRE site was found, and the results of EMSAs and dual luciferase assays provided evidence to show that CsARF5 could bind to the promoter of CsDREB3 and activate its expression (Figure 8).
Based on previous studies in model plant species and our results in this study, a working model of CsARF5 regulating the H 2 S-mediated cold stress response was proposed (Figure 9). CsIAA proteins interact with CsARF5 and interfere with the transcriptional regulation of CsDREB3 by CsARF5. When H 2 S signaling was detected, auxin levels in cucumber were increased, and CsARF5 was freed from the CsIAA protein complex. Then, CsARF5 specifically activated the expression of CsDREB3 to improve the cold tolerance of cucumber. Our study elucidates the molecular mechanism by which H 2 S regulates the cold stress response in cucumber by mediating auxin signaling, which will provide insights for further studies on the molecular mechanisms by which H 2 S regulates cold stress. A better understanding of the function and signal transduction of CsARF5 in cucumber is helpful to regulate the resistance of cucumber to low-temperature stress to obtain high-quality fruit. Figure 9. A regulatory model elucidates that H 2 S mediates the cold stress response in cucumber through auxin signaling. In the absence of H 2 S, IAA repressor proteins inhibit the expression of CsARF5, which in turn inhibits the cold stress response mediated by the CsARF5-CsDREB3 module. In the presence of H 2 S, IAA repressor proteins release CsARF5, which promotes the cold stress response by activating CsDREB3 expression.

Plant Material and Growth Conditions
'Jinyou 35' cucumber seedlings were used for cold stress treatment and genetic transformation. After soaking and germinating, the cucumber seeds were sown in nutrition bowls and transferred to a climate chamber with a PFD of 600 µmol m −2 ·s −1 , a 25 • C/16 • C thermo-period, an 11-h photoperiod and 80% relative humidity.

Vector Construction and Transient Transformation
To generate CsARF5 and CsDREB3 overexpression vectors, full-length CsARF5 and CsDREB3 were inserted into the pCAMBIA1300 plasmid.
For the transient transformation of detached cucumber leaves, cucumber leaves with the same growth conditions were taken, and Agrobacterium tumefaciens LBA4404 (Weidi, Shanghai, China) with overexpression vector was injected into cucumber leaves through a medical syringe.

Cold Stress Treatments
To evaluate the effect of H 2 S and IAA on the cold resistance of cucumber, cucumber seedlings with two leaves were foliar sprayed with 1.0 mM NaHS (an H 2 S donor; Shanghai Macklin Biochemical Co., Ltd., Shanghai, China), 0.15 mM HT (a specific scavenger of H 2 S; Sigma-Aldrich, Shanghai, China) and deionized water (H 2 O), respectively, or pretreated with 75 µM IAA (Solarbio, Beijing, China), 50 µM NPA (a polar transport inhibitor of IAA; Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China) or deionized water (H 2 O), respectively. Twenty-four hours later, half of the treatments were exposed to low temperatures (5 • C), and the other half of the seedlings were placed at normal temperatures as the control. The EL and accumulation of ROS were determined at 48 h after exposure of seedlings to 5 • C. Leaf samples of seedlings pretreated with H 2 O and 1.0 mM NaHS were collected from 3 plants (n = 3) for transcriptome analysis after 6 h of cold stress.
To compare the difference in cold tolerance between WT and transgenic cucumber leaves, the pCAMBIA1300 empty vectors and overexpressed CsARF5 and CsDREB3 vectors were injected into the first leaf, which was just flat of cucumber. Twelve hours later, WT leaves, CsDREB3 overexpressing leaves, and some CsARF5 overexpressing leaves were exposed to 5 • C. Other CsARF5 overexpressing leaves were treated with HT and then exposed to 5 • C after the water droplets on the leaves were absorbed and dried. The gene expression of CsDREB3, CsCBF1 and CsCOR in transgenic cucumber leaves was measured at 3 h after exposure to cold stress. NBT staining, DAB staining and ROS content were detected after cold treatment for 12 h.

qRT-PCR Analysis
The transcription levels of CsARF5, CsDREB3, CsCBF1 and CsCOR were examined using specific primers CsARF5 (qRT)-F/R, CsDREB3 (qRT)-F/R, CsCBF1 (qRT)-F/R and CsCOR (qRT)-F/R, respectively. β-Actin was used as an internal reference. All of the primers used are shown in Supplemental Table S1. qRT-PCR was carried out simultaneously with three biological replicates and three technical replicates.

Detection of EL
EL was detected according to methods described by Dong et al. (2013) [88]. Leaf discs (0.2 g) were immersed in 20 mL deionized water and incubated at 25 • C for 3 h. The electrical conductivity (EC1) was estimated using a conductivity meter (DDB-303A, Shanghai, China). The leaf discs were boiled for 10 min and then cooled to detect EC2. EL was calculated according to the following formula: EL= EC1/EC2 × 100.

IAA Content and FMO Activity Assay
IAA content was measured using high-performance liquid chromatography-triple quadrupole mass spectrometry (Thermo Fisher Scientific, TSQ Quantum Access, San Jose, CA, USA) according to the method of Li et al. (2014) [89], with minor modifications by Zhang et al. (2020) [60]. In brief, leaf samples were extracted with 80% methanol (containing 30 µg·mL −1 sodium diethyldithiocarbamate), and the supernatant was retained by rotary evaporation (Shanghai EYELA, N-1210B, Shanghai, China). Pigment and phenolic impurities of samples were removed using trichloromethane and polyvinylpolypyrrolidone (PVPP), respectively. Auxin was further extracted with ethyl acetate, and then the ester phase was collected. Finally, rotation drying at 36 • C and drying were dissolved in 1.0 mL mobile phases (methanol: 0.04% acetic acid = 45:55, v/v). The filtrate could then be used directly for HPLC-MS analysis.  [93]. The cleaned fresh leaves were soaked in 1 mM DAB staining solution (pH 3.8) in a vacuum and stained at 28°C for 8 h. Then, the leaves were boiled in ethanol:lactic acid:glycerol (3:1:1) mixed solution to remove pigments and H 2 O 2 was visualized in reddish-brown coloration.

EMSAs
The CsARF5-HIS fusion protein and biotin labeled probes were prepared for EMSAs. The CsARF5-HIS fusion protein was obtained by inducing Escherichia coli BL21 (TransGen Biotech, Beijing, China) with isopropyl β-D-thiogalactoside (IPTG). The biotin-labeled probes were synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. To perform the EMSAs, the fusion protein was mixed with the probe and incubated at 24 • C for 30 min. The protein-probe mixture was separated by nondenatured acrylamide gel electrophoresis.

Dual Luciferase Assay
The promoter sequence of CsDREB3 was amplified and cloned into pGreenII 0800-LUC to generate the reporter construct pCsDREB3-LUC. The effector plasmid was constructed by inserting full-length CsARF5 into pGreenII 62-SK. Different plasmid combinations were injected into tobacco (Nicotiana benthamiana) leaves by Agrobacterium tumefaciens LBA4404. The leaves were sprayed with 100 mM luciferin, and luminescence was detected after being placed in darkness for 3 min. Fluorescence images were obtained with a live imaging system (Xenogen, Alameda, California, USA). The fluorescence activity was determined using a fluorescence activity detection kit (Promega, Madison, WI, USA).

Statistical Analysis
All experiments were performed at least in triplicate, and the results are expressed as the mean ± standard deviation (SD) of three replicates. The data were analyzed statistically with DPS software. Duncan's multiple range test was used to compare differences among treatments, and p < 0.05 was considered statistically significant.

Conclusions
H 2 S treatment increases cold tolerance and auxin content of cucumber. The auxin response factor CsARF5 directly activates the expression of CsDREB3 to improve the cold tolerance of cucumber in response to H 2 S treatment. This study elucidates the molecular mechanism by which H 2 S regulates the cold stress response in cucumber by mediating auxin signaling, which will provide insights for further studies on the molecular mechanisms by which H 2 S regulates cold stress.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ijms222413229/s1. Author Contributions: X.A. and X.Z. designed the experiment; X.Z. performed the research and analyzed the data; X.F., F.L., Y.W. and H.B. worked together with X.Z. to accomplish the experiment X.A. and X.Z. wrote the paper. All authors have read and agreed to the published version of the manuscript.