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Plant Molecular Biology (2019) 99:535–544 https://doi.org/10.1007/s11103-019-00834-w

The role of  H2S in low temperature-induced cucurbitacin C increases in cucumber

Zhiqiang Liu1 · Yawen Li1 · Chunyu Cao1 · Shan Liang2,3 · Yongshuo Ma4 · Xin Liu5 · Yanxi Pei1

Received: 20 November 2018 / Accepted: 26 January 2019 / Published online: 1 February 2019 © Springer Nature B.V. 2019

AbstractKey message In this study, we first linked the signal molecule H2S with cucurbitacin C, which can cause the bitter taste of cucumber leaves and fruit, and specifically discuss its molecular mechanism.Abstract Cucurbitacin C (CuC), a triterpenoid secondary metabolite, enhances the resistance of cucumber plants to patho-genic bacteria and insect herbivores, but results in bitter-tasting fruits. CuC can be induced in some varieties of cucumber on exposure to plant stressors. The gasotransmitter hydrogen sulfide (H2S) participates in multiple physiological processes relating to plant stress resistance. This study focused on the effect of H2S on low temperature-induced CuC synthesis in cucumber. The results showed that treatment of cucumber leaves at 4 °C for 12 h enhanced the content and production rate of H2S and increased the expression of genes encoding enzymes involved in H2S generation, Csa2G034800.1 (CsaLCD), Csa1G574800.1 (CsaDES1), and Csa1G574810.1 (CsaDES2). In addition, treatment at 4 °C or with exogenous H2S upregu-lated the expression of CuC synthetase-encoding genes and the resulting CuC content in cucumber leaves, whereas pre-treatment with hypotaurine (HT, a H2S scavenger) before treatment at 4 °C offset these effects. In vitro, H2S could increase the S-sulfhydration level of His-Csa5G156220 and His-Csa5G157230 (both bHLH transcription factors), as well as their binding activity to the promoter of Csa6G088690, which encodes the key synthetase for CuC generation. H2S pretreatment enhanced the cucumber leaves resistance to the Phytophthora melonis. Together, these results demonstrated that H2S acts as a positive regulator of CuC synthesis as a result of the modification of proteins by S-sulfhydration, also providing indirect evidence for the role of H2S in improving the resistance of plants to abiotic stresses and biotic stresses by regulating the synthesis of secondary metabolites.

Keywords Cucurbitacin C · Hydrogen sulfide · Low temperature stress · S-sulfhydration · Phytophthora melonis

Introduction

Cucumber (Cucumis sativus L), one of the most important crops in the Cucurbitaceae family, is widely grown around the world. Cucumber fruits are both edible and beneficial to human health because they are rich in vitamin A, vitamin C, minerals and micronutrients. Extracts from cucumbers show anti-inflammatory effects, relieving both the production of phlegm and muscular spasms, as recorded in both Ben cao gang mu and The Flora of China. Cucurbitacins are highly oxygenated tetracyclic triterpenoids that are synthesized in cucurbitaceous plants as antiherbivore defenses (Da and Jones 1971), and might also be responsible for the phar-macological properties described above (Chen et al. 2005). Cucurbitacins B and E, which are ubiquitous in nature and considered to be the original form of cucurbitacin (Ling et al. 2010), have been used as adjuvant treatments for hepatitis

Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1110 3-019-00834 -w) contains supplementary material, which is available to authorized users.

* Yanxi Pei peiyanxi@sxu.edu.cn

1 School of Life Science, Shanxi University, Taiyuan 030006, China

2 Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology and Business University, Beijing 100000, China

3 Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing 100000, China

4 Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518124, China

5 School of Life Sciences, Qingdao Agricultural University, Qingdao 266109, China

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and primary liver cancer (Ling et al. 2010; Jayaprakasam et al. 2003). Thus, research has begun to focus on these com-pounds in terms of their medicinal effects.

In cucumber, the major bitter compound is cucurbitacin C (CuC), which is synthesized quickly in response to herbivory and environmental stresses, such as low (< 13 °C) or high temperature (> 30 °C), insufficient light intensity, exces-sive or insufficient nitrogen, and soil drought (Zhang et al. 2011), resulting in bitter-tasting fruits in certain varieties of cucumber (Li et al. 2015; Kano and Goto 2002). Thus, cultivated cucumber can be classified into three categories according to the generation of bitter taste. Varieties in the first category have bitter-tasting vegetative organs and fruits, which increase the bitterness when the plants are grown in unsuitable environments. Most of the cucumbers grown in southern and northern China fall into this category. In the second category, the vegetative organs are bitter, whereas the fruits do not develop any bitterness. This is because the fruits contain a pair of dominant genes that encode proteins that inhibit the formation of CuC. Most American and some Northern China cucumber varieties belong to this category. Neither vegetative organs nor fruits of plants in the third category are bitter, such as varieties grown in The Nether-lands (Gu et al. 2004). Considering the edible and resistant qualities of CuC on cucumber, and the medicinal effects, it is meaningful to study the regulatory mechanism of CuC synthesis.

The genes controlling bitterness in cucumber are the Bi (Bitter) and Bt (Bitter fruit) genes, whereby Bi genes control CuC synthesis throughout the plant, whereas both Bi and Bt genes control CuC synthesis in the fruit. Huang et al. identified nine genes encoding enzymes involved in CuC biosynthesis, including Csa6G088690, which encodes 2,3-oxidosqualene cyclase, which catalyzes the formation of the cucurbitane skeleton, the first, key step in the genera-tion of CuC. Other genes include cytochrome P450-encod-ing genes (Csa6G088160, Csa6G088170, Csa3G698490, Csa1G044890, Csa6G088710, Csa3G903540, and Csa3G903550) and an acetyltransferase C-encoding gene (Csa6G088700), the products of which are involved in catalyzing the modification of the cucurbitane skeleton resulting in the synthesis of CuC (Shang et al. 2014; Zhou et al. 2016a, b). Two bHLH transcription factor-encoding genes (Csa5G156220 and Csa5G157230), expressed spe-cifically in leaves and fruit, respectively, combine with the promoters of Bi genes to promote their transcription, result-ing in the accumulation of CuC in leaves or fruits. Thus, Csa5G156220 has been identified as a Bl (Bitter leaf) gene, whereas Csa5G157230 was identified as a Bt gene.

The gasotransmitter hydrogen sulfide (H2S) not only has important physiological functions in regulating plant devel-opment, such as delaying senescence, and promoting seed germination and root morphogenesis (Pei 2016), but also

enhances tolerance against multiple stresses, such as induc-ing Arabidopsis stoma closure to improve drought resist-ance (Jin et al. 2011), improving the activity of superoxide dismutase (SOD), catalase (CAT) and antioxidant molecules to avoid the accumulation of reactive oxygen species (ROS), and alleviating oxidative damage caused by heavy metal stress (Tian et al. 2016; Fang et al. 2014) or chilling stress (Wang et al. 2015). Cucumber is a cold temperature-sensi-tive plant, and H2S alleviates damage to cucumber leaves caused by low temperatures by increasing the degree of PSII reaction center opening, maintaining high carbon assimila-tion capacity and electron transport rates (Zhou et al. 2016a, b). The physiological concentrations of H2S also enhance the ability of plants to resist biotic and abiotic stresses by regulating the synthesis of secondary metabolites (Chen et al. 2016; Shi et al. 2015). For example, H2S can promote the synthesis of nicotine in tobacco under high temperature conditions by regulating the transcription of genes involved in the biosynthesis of jasmonic acid. In addition, H2S can increase the content of polyphenol, flavonoid and triterpe-noid secondary metabolites in birch suspension cells (Wang et al. 2017). However, there has been no report of an associa-tion between H2S and CuC, an unstable secondary metabo-lite that controls the bitter taste of cucumber fruits and helps cucumber plants to resist herbivory and bacterial invasion.

Cucumber phytophthora blight is the main disease caused by Phytophthora melonis, which often causes the death of a large number of plants and rotten melon (Zhou et al. 2015). During the whole development, all parts can be affected at various stages. The damage parts are mainly cucumber fruit, leaves and stems, and the base of stem is especially serious, which seriously affects the growth of cucumber. Since CuC has the special physiological functions mentioned above, and it has been documented that CuC can inhibit the growth of Phytophthora cactorum (Nes and Patterson 1981). There-fore, given the anticancer, anti-inflammatory and other phar-macological effects of CuC, we focused on the molecular mechanism of H2S in regulating CuC synthesis in cucum-bers subjected to chilling stress, with the aim of providing a theoretical basis for H2S to enhance the biological resistance of plants by regulating secondary metabolites. Meanwhile, it also provides a new idea for the breeding of cucumber cultivar and the prevention and control of Cucumber phy-tophthora blight.

Results

Both H2S content and production rate increased in cucumber leaves under 4 °C

H2S is produced in response to multiple environmental stresses in plants, such as in Vitis vinifera L or cucumber

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‘Jinyou 35’ under chilling stress. To confirm the universality of H2S signal activation in cucumber under chilling stress, four cucumber varieties (XY-3, XY-2, 9930, and han) were kept at 4 °C for 12 h, and the H2S content of the leaves were determined. As shown in Fig. 1A, the endogenic H2S content increased significantly in each of the four cucumber lines in plants exposed to 4 °C, but not in seedlings pretreated with HT for 24 h before chilling stress. LCD, the activity of which is reflected by the H2S production rate, is reported as the major pathway for H2S generation in plants; in the current study, the activity of LCD was detected through the methylene blue method. The protein extracted from cucum-ber leaves was used to explore the effect of chilling stress on LCD/H2S. As shown in Fig. 1B, H2S production rates increased in cucumbers exposed to 4 °C, whereas HT pre-treatment inhibited the activity of LCD in 9930 and han.

LCD and DES gene expression was upregulated in cucumber leaves under 4 °C

Two genes encoding the major H2S-generating enzymes in Arabidopsis, LCD (At3G62130) and DES (At5G28020). In cucumber, the amino acid sequence encoded by Csa2G034800.1 is most similar to that of AtLCD, with a 60% amino acid identity and was named CsaLCD (Supplementary Fig. S1). Both Csa1G574800.1 and Csa1G574810.1 encode AtDES homologous proteins, with 60% (Supplementary Fig.

S2A) and 69% (Supplementary Fig. S2B) amino acid iden-tity, respectively, and were named CsaDES1 and CsaDES2. Csa1G574810.1 showed almost exactly the same sequence identity as the C terminus of Csa1G574800.1 (Supplemen-tary Fig. S3). The relative transcription levels of CsaLCD, CsaDES1, and CsaDES2 in cucumber leaves under chilling stress were detected by RT-qPCR. As shown in Fig. 2A, the mRNA level of CsaLCD was induced in 9930, XY-3, XY-2 and han by 4 °C treatment, whereas CsaDES1 was induced in 9930, and CsaDES2 was induced in 9930, XY-2, and han. The levels of CsaLCD protein in the four cucumber varie-ties were also detected by western blot, and the results were concordant with the mRNA level induced by chilling stress (Fig. 2B).

H2S is involved in increasing low temperature‑induced CuC synthetase gene expression in cucumber

The expression of the CuC synthetase genes in multi-ple organs of four cucumber varieties was determined to reveal whether H2S signals participate in chilling stress-induced CuC synthesis in cucumber. The expres-sion of Csa6G088690, Csa6G088700, Csa6G088160, Csa6G088170, Csa3G698490, and Csa1G044890 was upregulated in leaves of 9930 and XY-2 following 4 °C treat-ment, but was partly reduced by HT pretreatment before

Fig. 1 Low temperature-induced endogenic H2S generation in cucumber leaves. Three-leaf-stage cucumber seedlings were treated at 4  °C for 12 h (4 °C) or sprayed with 200 µmol/L hypotaurine three times over 24 h and then treated at 4 °C for 12 h (4 °C + HT); HT rep-resents three-leaf stage seedling were sprayed with 200  µmol/L HT three times over 24 h, after pretreatment, normal growth at 23 °C for

12 h; CK represents the untreated control. The H2S content of leaves was detected by specialized electrodes (A), and the H2S production rate was detected by the methylene blue method (B). Data represent the mean ± SE of three independent replicates. Bars with different letters (a, b, ab) indicate statistically significant differences (Tukey’s multiple range test, P < 0.05)

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chilling stress (Fig.  3). There was no difference in the expression of CuC synthetase-encoding genes in leaves of XY-3 and han subject to 4 °C treatment or to HT pretreat-ment before chilling (Fig. 3). The upregulated expression of genes in 9930 and XY-2 following 4 °C treatment was also recorded in cotyledons (Supplementary Fig. S4A), stems (Supplementary Fig. S4B) and pedicels (Supplementary Fig. S4C) following exogenous H2S treatment. No increases were recorded in XY-3 (Supplementary Fig. S4A–C), in fruits of 9930 or in han (Supplementary Fig. S4D) following exog-enous H2S treatment.

H2S induces the expression of CuC‑encoding genes by S‑sulfhydration of transcription factors

The expression of CuC synthetase-encoding genes is reg-ulated by two bHLH transcription factors (Csa5G156220 and Csa5G157230), expressed in leaves and fruit, respec-tively (Shang and Huang 2015). The current results showed that exogenous H2S induced the expression of CuC syn-thetase genes (Supplementary Fig. S4) and, thus, that H2S signals were involved in upregulating the expression of chilling stress-induced CuC synthetase-encoding genes. Recent studies showed that H2S increases protein activ-ity by S-sulfhydration, a post-translational modification

Fig. 2 Expression of genes encoding enzymes involved in H2S gen-eration induced in cucumber leaves under 4  °C. Three-leaf-stage cucumber seedlings were treated at 4  °C for 12  h (4  °C); CK rep-resents the untreated control. A The relative transcription levels of CsaLCD and CsaDES in cucumber leaves were detected by RT-qPCR. Data represent the mean ± SE of three independent replicates. Bars with ‘*’ indicate statistically significant differences (Tukey’s multiple range test, P < 0.05). B Immunoblot detection of CsaLCD in cucumber XY-3, XY-2, 9930 or han varieties with LCD antibodies. CsaActin was used as a loading control

Fig. 3 Effects of H2S on the expression of CuC synthetase genes in cucumber leaves under 4  °C. Three-leaf-stage cucumber seedlings were treated at 4  °C for 12  h (4  °C) or sprayed with 200  µmol/L hypotaurine three times over 24 h and then treated at 4 °C for 12 h (4  °C + HT); HT represents three-leaf stage seedling were sprayed with 200 µmol/L HT three times over 24 h, after pretreatment, nor-

mal growth at 23  °C for 12  h; CK represents the untreated control. The relative expression of CuC synthetase-encoding genes in cucum-ber leaves was detected by RT-qPCR. The 2−δδCt method was used to calculate the relative mRNA levels of the genes. Data represent the mean ± SE of three independent replicates

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of Cys residues of proteins (Ju et al. 2017). Thus, recom-binant His-Csa5G156220 or His-Csa5G157230 protein purified from E. coli were treated with 10 µmol/L H2S, and then their S-sulfhydration levels were detected by a biotin switch assay. The results showed that H2S treatment increased the sulfhydration levels of His-Csa5G156220 and His-Csa5G157230 (Fig.  4A). Shang reported that Csa5G156220 and Csa5G157230 bind to the promot-ers of the CuC synthetase-encoding genes Csa6G088690, Csa6G088160 and that Csa6G088690 catalyzes the first, key

step in the generation of CuC. Our GSA experiments dem-onstrated that H2S treatment increased the binding activity of Csa5G156220 and Csa5G157230 to the Csa6G088690 promoter probe fragment (Fig. 4B).

CuC content is increased by exogenous H2S treatment

The results showed that the CuC content increased under low temperature stress and that H2S promoted the binding activity of Csa5G156220 and Csa5G157230 to the promoter of Csa6G088690 via sulfhydration modification of two tran-scription factors, inducing the expression of CuC synthetase-encoding genes. The CuC content of different organs of 9930 were detected by HPLC. The CuC content of leaves, cotyle-dons, pedicel, carpopodium and fruit increased significantly after 4 °C or exogenous H2S treatments in cultivar 9930, but these increases were partly prevented by HT pretreatment before exposure to 4 °C (Table 1).

Exogenous H2S can significantly improve the resistance of cucumber leaves to Phytophthora melonis

H2S can increase the content of CuC, and the increase of CuC can help plants resist the infection of pathogenic bac-teria. To confirm whether H2S is involved in the process of cucumber leaf resistance to phytophthora blight. The cucumber leaves inoculated with Phytophthora melonis and then the growth state of Phytophthora melonis plaques was observed. After 48 h of infection, there was no significant change in the plaque of Phytophthora melonis in the three groups. Cucumber leaves were pretreated with NaHS (H2S donor), the diameter of Phytophthora melonis plaque was significantly lower than the other two groups at 72 h; 96 h was particularly obvious (Fig. 5). The above results indicate that H2S can significantly improve the resistance of cucum-ber leaves to phytophthora blight.

Fig. 4 H2S treatment increased the sulfhydration levels and bind-ing activity of the transcription factors (TFs) Csa5G156220 and Csa5G157230. A Purified His-Csa5G156220 and His-Csa5G157230 proteins were treated with 10 µmol/L H2S for 30 min, and the sulfhy-dration levels were detected by biotin switch assay and western blots with anti-biotin antibodies. B GSA showed that the binding activ-ity of Csa5G156220 and Csa5G157230 TFs to the Csa6G088690 promoter was enhanced by H2S treatment. The fusion proteins were treated with 5 or 10  µmol/L H2S for 30  min, respectively, before GSA, and the E-box-containing promoter fragment of the Csa6G088690 gene was labeled with digoxigenin-dUTP

Table 1 Comparison of the CuC content of different tissues from 9930

The 9930 variety was fumigated with 50 µmol/L H2S for 12 h, treated at 4 °C for 12 h (4 °C), or sprayed with 200  µmol/L hypotaurine three times over 24  h and then treated at 4  °C for 12  h (4  °C + HT); CK represents the untreated control. The CuC content of different tissues were detected by HPLC. Values are the means ± standard deviation (n = 3). Different letters (a, b, c) indicate statistically significant differences (Tukey’s multiple range test, P < 0.05)

Leaves (µg/g FW) Cotyledon (µg/g FW) Pedicel (µg/g FW) Carpopo-dium (µg/g FW)

Fruit (µg/g FW)

CK 10.1 ± 3.2a 19.3 ± 4.1a 12.9 ± 4.6a 14 ± 3.7a 10.4 ± 2.4a

H2S 32.2 ± 6.4c 46.7 ± 5.8c 24.1 ± 5.2b 38.8 ± 4.2b 37.7 ± 5.6c

4 °C 34.6 ± 5.9c 36.2 ± 4.7b 27.9 ± 4.4b 35.4 ± 5.5b 26.6 ± 4.8b

4 °C + HT 26.1 ± 3.8b 22.1 ± 3.6a 24.2 ± 4.1b 16.2 ± 3.4a 11.8 ± 3.3a

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Discussion

The gasotransmitter H2S enables plants to resist multiple abiotic stresses, such as drought, salt, heavy metals, unsuit-able temperatures, by activating antioxidant systems and regulating the expression of stress-related genes (Cui and Shen 2012; Wang 2012); however, the related reports of H2S on plant biotic stress resistance are limited. Secondary metabolites, generated through secondary metabolism and always nonessential for plant cell activity or plant growth and development under normal conditions, help plants to resist diseases or pests and are significant for human beings; examples include artemisinin (Yin et al. 2014) and cucurbitacin (Chambliss and Jones 1966). Cucurbitacin, a highly oxidized tetracyclic triterpenoid, is a characteristic

compound in more than 100 species from more than 30 gen-era of Cucurbitaceae, and protects the plant from numerous herbivores and pathogens. In cucumber, the major cucurbi-tacin is CuC, which is induced by multiple abiotic stresses and insect herbivory. For example, increased CuC synthesis reduces injuries caused by Tetranychus urtica or Aulacoph-ora femoralis chinensi. However, CuC can result in bitter-tasting cucumber fruits. H2S signals promote the synthesis of secondary compounds in plants, such as nicotine, and both H2S and CuC are quickly induced when cucumbers are exposed to low temperatures (< 13 °C) (Wu et al. 2017; Chen 2015). In the current study, the results suggested that H2S signals were induced by chilling stress in cucumber and that HT was an effective scavenger of endogenic H2S (Fig. 1); H2S had a positive role in inducing CuC synthesis

Fig. 5 Effect of exogenous H2S on cucumber leaves resistance to Phytophthora melonis. Three-leaf-stage cucumber seedlings were fumigated with 50  µmol/L NaHS (H2S donor) or sprayed with 200  µmol/L HT (H2S scavenger) for 12  h, The CK group was pre-treated with water for 12 h as a control. The diameter of 5 mm Phy-

tophthora melonis plaque was inoculated on leaves and the plaque diameter was measured every 24 h. Data represent the mean ± SE of three independent replicates. Bars with ‘*’ indicate statistically sig-nificant differences (Tukey’s multiple range test, P < 0.05)

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(Table 1), providing evidence for H2S signals strengthening plant biotic stress resistance. The concentration of CuC was unstable in different plant organs, and was more abundant in younger parts of the plant. It is difficult to separate and purify large amounts of CuC, which limits its pharmaceuti-cal applications; however, this could change in the future following studies on the H2S signal induction pathway.

The regulation of gene expression is one of a range of physiological effects of H2S in plant resistance to stresses. For example, H2S can enhance the tolerance of millet to heavy metals by regulating the expression of heavy metal chelator-associated genes (Fang et  al. 2014) and metal transporter genes (Tian et al. 2017). H2S also responds to drought stress by regulating drought-associated miRNAs and drought-responsive genes (Shen et al. 2013). Accord-ing to Shang et al. (2014), the CuC synthetase-encoding genes Csa6G088690, Csa6G088700, Csa6G088160, Csa6G088170, Csa3G698490, and Csa1G044890 are regulated by the bHLH transcription factors Csa5G156220 and Csa5G157230, whereas Csa6G088180 encodes a cytochrome P450 but is not regulated by either of the two transcription factors. Csa6G088690 encodes 2,3-oxidos-qualene cyclase, which catalyzes the first, key step in the generation of CuC. H2S enhanced the binding activity of His-Csa5G156220 and His-Csa5G157230 proteins to the Csa6G088690 gene promoter in vitro via S-sulfhydration (Fig. 4), and induced the expression of Csa6G088690 and other CuC synthetase-coding genes (Fig. 3). Sulfhydration of transcription factors is a common means by which H2S signals regulate gene expression, but more evidence in vivo is needed. In addition, more evidence is required in terms of protein activity before and after sulfhydration, particularly in the form of crystal structures and bioinformatics data.

At present, the main measures to control cucumber phy-tophthora blight are chemical control and biological con-trol. Chemical control seriously endangers the ecological environment. Therefore, the use of beneficial microorgan-isms and their metabolites to control cucumber blight is the main direction of modern research. The bacteria that have been used to prevent and control cucumber phytophthora blight are Bacillus subtilis (Wu et al. 2014) and Strepto-myces purpeofuscus (Zhou et al. 2015). The application of secondary metabolites to the prevention of cucumber blight has high research value. As a gas signal molecule that can participate in a variety of stress responses, H2S can improve the ability of grape to resist Plasmopara viticola (Wang et al. 2013). In this study, we have shown that H2S is involved in the regulation of CuC content (Table 1), which the increase can resist the damage of Phytophthora blight. we also found that H2S is also directly involved in the process of cucum-ber resistance to Phytophthora blight (Fig. 5). The expres-sion levels of L-type lectin receptor kinase 6.1 (LecRK6.1), glucanase and chitinase were examined, which three basic

defense-related transcripts play an important role in plant defense responses to pathogen infection (Wu et al. 2014, Ramezani et al. 2018). After infected by Phytophthora melo-nis, The mRNA levels of LecRK6.1 and chitinases increased significantly at 24 h and 48 h, while the mRNA levels of CuC synthetase genes increased slightly (Supplementary Fig. S7). Exogenous H2S treatment can also induce the expression of defense-related genes and CuC synthetase genes (Supplementary Fig. S6). These results provide evi-dence that H2S increase CuC against Phytophthora melonis. This is only a new idea to provide H2S resistance to Phy-tophthora melonis infection, and more pathways still require further investigation.

In summary, there have been many reports on H2S signal help plants resist stresses and the fact that CuC content of the variety (Table 1) and the Phytophthora melonis infesta-tion on the leaves (Fig. 5) are detected. This study identi-fied that the sulfhydration modification on two transcription factors by H2S increases the transcriptional activity of CuC synthetase, which in turn increases the content of CuC. At the same time, since CuC can also enhance the ability of plants to resist biotic stress, it provides new evidence for H2S to respond to biotic stress through the content of secondary metabolites.

Materials and methods

Plant materials and treatments

Four cucumber varieties were used in this study: 9930 (bit-terness inducible in fruits and leaves); han (bitterness unin-ducible in fruits); XY-2 (bitterness inducible in leaves only); and XY-3 (bitterness uninducible in leaves). The seeds were soaked in ddH2O for 24 h and then grown in pots containing a soil:perlite:vermiculite [1:1:1(v/v)] mixture. The pots were kept at 23 °C, 60% relative humidity (RH), and in a 16 h/8 h (light/dark) photoperiod with light illumination at 160 µE/m2/s. Three-leaf-stage seedlings were stressed at 4 °C for 12 h, or sprayed with 200 µmol/L hypotaurine (HT, a H2S scavenger) three times over 24 h, followed by 4 °C treatment for 12 h. The second leaf from top to bottom were then taken to determine the H2S production rate, H2S content, and rel-evant gene expression.

Three-leaf-stage seedlings were sprayed with 100, 200, 300 µmol/L HT three times over 24 h, the second leaf from top to bottom were then taken to determine the content of soluble protein, malondialdehyde (MDA) and soluble sugar.

Cucumber plants from different growth stages were selected for exogenous H2S treatment, 50 µmol/L of H2S (NaHS as an exogenous H2S donor) were fumigated for 12 h. The expression level of CuC synthetase-coding genes in different parts was determined, such as freshly opened

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cotyledons (germination period), stems at the three-leaf-stage, pedicel before flowering (initial bloom stage), and fruits (initial bloom stage). CK as an internal reference. Samples from different parts were ground to a powder form with liquid nitrogen and stored in a − 80 °C refrigerator for subsequent determination of related gene expression.

Cucumber plants from different growth stages were selected for stress treatment, different parts were selected for determination of CuC content, such as freshly opened cotyledons (germination period), stems at the three-leaf stage, stalks before flowering (initial bloom stage), carpo-podium (initial bloom stage) and fruits (initial bloom stage). All cucumber plants were divided into four groups. The first group was a normally grown plant as a blank control. The second group was fumigated with 50 µmol/L of exogenous H2S (NaHS as an exogenous H2S donor) for 12 h. The third group was treated with 4 °C for 12 h. The fourth group was sprayed with 200 µmol/L HT three times over 24 h, followed by 4 °C treatment for 12 h. The other three groups of pre-treatment are sprayed with water. Samples of different parts were ground into powder with liquid nitrogen and stored in a − 80 °C refrigerator for subsequent determination of CuC content.

Determination of soluble protein content, MDA and soluble sugar content

0.2–0.3 g of leaves with various treatments was ground in 1 mL PBS buffer (0.05 M, pH 7.0) and the homogenate was centrifuged, 12,000 rpm/min for 15 min at 4 °C, then extract the supernatant of 900µL for the determination of the above physiological indexes.

Determination of MDA content by thiobarbituric acid (TBA) method (Aghdama et al. 2018). 0.2 mL of superna-tant was mixed with 0.2 mL of 0.67% TBA in a centrifuge tube and boiled in water at 100 °C for 15 min. The solu-tion was centrifuged at 3000 rpm/min for 5 min at 4 °C and then measured the absorbance of the supernatant at 450 nm, 532 nm, 600 nm, respectively.

The content of MDA was calculated based on a standard curve of known concentrations of MDA.

Coomassie blue staining was used for the determination of soluble protein content. 10 µL of the supernatant was mixed with 390 µL of distilled water and 100 µL of Coomas-sie Brilliant Blue solution in a centrifuge tube, shaken, placed for 2 min to complete the reaction, and colorimetri-cally measured at 595 nm to determine the absorbance. The content of soluble protein was calculated based on a standard curve of known concentrations of protein.

Pipette 0.5 mL of the above sample extract in a 20 mL test tube, add 1.5 mL of distilled water, add 0.5 mL of anthrone reagent and 5 mL of concentrated sulfuric acid in order, shake well, immediately place the test tube in a boiling water

bath, and keep the tube for 1 min. After removal, it was naturally cooled to room temperature. The absorbance at 630 nm was measured, and the amount of sugar contained was determined from the standard curve. The content of soluble sugar was calculated based on a standard curve of known concentrations of sugar.

Determination of  H2S production rate

Total l-cysteine desulfhydrase activity was ascertained by measuring the H2S production rate from l-cysteine (Rie-menschneider et al. 2005). Total protein was extracted from leaves as follows: 0.3 g of fresh leaves was ground in 1 mL PBS buffer (0.05 M, pH 7.0) on ice and the homogenate was centrifuged. The supernatant was applied to determine the H2S production rate using the methylene blue method (Qiao et al. 2015), resulting in a production rate in nanomoles H2S per milligram protein per minute (Aghdam et al. 2018).

Determination of  H2S content

0.1 g of fresh leaves was ground in 1 mL PBS (0.05 M, pH 7.0, 0.2 M ASA, 0.1 M EDTA) buffer on ice. The H2S content was then determined using a specialized electrode (TBR4100 WPI, Sarasota, FL, USA) and calculated accord-ing to the standard curve of H2S content (Papenbrock and A 2007).

Extraction of total RNA and RT‑qPCR

Total RNA was isolated from 0.1 g fresh leaves using the RNAiso plus reagent (TaKaRa, Shiga, Japan, Cat 9109) according to the manufacturer’s instructions. The relative changes in the mRNA of target genes were detected by RT-qPCR according to the instructions of the Bio-Rad Real-Time System CFX96TM and calculated by using the 2−δδCt method. ACTIN was used as an internal control. The primer pairs are shown in Table S1.

Western blot assays

Cucumber leaves were ground into a fine powder in liq-uid nitrogen, and the protein was extracted in PBS buffer (0.05 M, pH 7.0). The extracted proteins were separated by SDS-polyacrylamide gel electrophoresis, and then trans-ferred to nitrocellulose membranes. Immunodetection was performed with a primary antibody of L-cysteine desulfhy-drase (LCD) (Biology Institute of Shanxi, China) and alka-line phosphatase conjugate goat anti-mouse IgG secondary antibodies, followed by visualization with BCIP-NBT buffer.

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Determination of CuC content

CuC was extracted from different parts of the cucumber plants by using methanol, and HPLC was used to determine the CuC content, as described previously (Kong et al. 2004).

Protein S‑sulfhydration assay

Recombinant His-Csa5G156220 and His-Csa5G157230 proteins were produced and purified from Escherichia coli according to the method of Shang et al. (2014), and then used for the S-sulfhydration assays. The purified proteins were precipitated by precooled acetone and redissolved in 100 µL HENS buffer (250 mM Hepes-NaOH (pH 7.7), 1 mM EDTA, 0.1 mM neocuproine and 1% SDS). After incubation with 10 µM H2S (NaHS as an exogenous H2S donor) for 30 min at 4  °C, the S-sulfhydration level of the proteins was detected by using a Biotin switch assay, as described previously (Mustafa et al. 2009). The Biotin antibody (Sigma-Aldrich, China) was used as the primary antibody in the western blots.

Gel‑shift assay (GSA)

The recombinant His-Csa5G156220 and His-Csa5G157230 proteins purified from E. coli were treated with 5 or 10 µmol/L H2S (NaHS as an exogenous H2S donor), respec-tively, and then used for the gel-shift assays (GSAs). The promoter fragments used for the GSA were amplified by PCR using the following primer pairs: forward primer 5′-CAT AAT AGT TAT CAC ATG CAAAG-3′, reverse primer 5′-CTA CCA CAC TTA TAT ATA AATC-3′. The promoter fragment was labeled at the base T with digoxigenin-dUTP (Roche, Mannheim, Germany) according to the manufac-turer’s instructions. Binding reactions and GSA were per-formed as described elsewhere (Shang et al. 2014).

Phytophthora melonis inoculation treatment

The three-leaf stage seedlings were divided into three groups for stress treatment, 50 µmol/L NaHS (H2S donor) fumiga-tion for 12 h, and 200 µmol/L HT (H2S scavenger) treatment for 12 h. In each of the three groups, The Phytophthora mel-onis plaque was inoculated on the leaves of a uniform leaf width with a punch having a diameter of 5 mm, then placed in 25 °C, and the growth diameter of the Phytophthora melo-nis plaque was measured every 24 h. Samples from different treatment times were ground to a powder form with liquid nitrogen and stored in a − 80 °C refrigerator for subsequent determination of related gene expression.

Statistical analysis

Each experiment was replicated three times. Values are expressed as means ± SE. Data were analyzed using SPSS (version 17, IBM SPSS, Chicago, IL), and error bars were calculated according to Tukey’s multiple range test (P < 0.05).

Acknowledgements We thank Dr. Sanwen Huang for Cucumber seeds. We also thank assistant researcher Jianbo Zhou for providing Phytoph-thora melonis strains. This work was supported by grants from the National Natural Science Foundation (Grant 31671605), and the pro-jects of Beijing Technology and Business University Youth Fund (No. QNJJ2017-06). We also thanks International Science Editing (http://www.inter natio nalsc ience editi ng.com) for editing this manuscript.

Author contributions ZL and YL have contributed equally to this work. ZL designed experiments; YL carried out experiments; ZL and YL analyzed experimental results. CC analyzed sequencing date and developed analysis tools. YM, SL and XL participated in the discussion and gave unique insights. YP reviewed and examined the experiment.

Funding This work was supported by grants from the National Natu-ral Science Foundation (Grant 31671605), and the projects of Beijing Technology and Business University Youth Fund (No. QNJJ2017-06).

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

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