The role and proteomic analysis of ethylene in hydrogen gas-induced adventitious rooting development in cucumber (Cucumis sativus L.) explants

Plant materials and treatments

Cucumber (Cucumis sativus L. var. ‘Xinchun 4’) seeds were purchased from Gansu Agricultural Institute (Lanzhou, China). The seeds were surface-sterilized in 5% sodium hypochlorite for 10 min and washed with water. The seeds were germinated on filter paper with distilled water in Petri dishes (15 cm-diameter, 2.5 cm-deep) and maintained at 25 ± 1° C for 7 d with a 14-h photoperiod (photosynthetically active radiation = 200 µmol m⁻²s⁻¹). The 7-day-old seedlings with primary roots removed were used as explants and were maintained under the same temperature and photoperiod conditions for another 7 days in the presence of different media as indicated below. We described these different treatments as (I) HRW (0, 1%, 10%, 50% and 100%); (II) ethephon (0, 0. 1, 0. 5, 1, 10 and 50 µmol  L⁻¹); (III) AVG (aminoethoxyvinylglycine, 0, 0. 1, 1, 5 and 10 µmol  L⁻¹); (IV) AgNO₃ and NaNO₃ (0, 0. 1, 0. 3 and 0. 5 µmol L⁻¹); and (V) 50% HRW + 0. 5 µmol L⁻¹ ethephon, 1 µmol  L⁻¹AVG, or 0. 1 µmol  L⁻¹ AgNO₃. After applying treatment explants were sampled to determine root number per explants. The hypocotyls (5-mm-long segments of the hypocotyl base where the adventitious root develops) of the explants after 48 h of treatment were immediately used or frozen in liquid nitrogen for further analysis.

Preparation of hydrogen-rich water (HRW)

Purified H₂ gas (99. 99%, v/v) generated from a hydrogen gas generator (QL-300, Saikesaisi Hydrogen Energy Co., Ltd., China) was bubbled into distilled water at a rate of 300 mL min⁻¹ for 30 min. The corresponding HRW was rapidly diluted to the required saturations (Zhu et al., 2016). The H₂ concentration analyzed by gas chromatography in freshly prepared HRW was 0. 68 mM, which was defined as 100% HRW, and was maintained at a relatively constant level in 25 °C for at least 12 h.

Quantification of ethylene

ETH was detected by a gas chromatographic analyzer (SP-3420A, Beifenruili Inc., China). Five fresh cucumber explants were placed in 20 mL vials, stoppered with secure rubber caps, and containing 1 mL culture solution. These vials had the same conditions as before when the seedlings grew for 12 h. A gas sample (2.5 mL) was extracted and injected into a gas chromatograph, which was fitted with a GDX-502 column (2 m × 3.2 mm) and a flame ionization detector. The temperature of the column, inlet and detector were 50, 140 and 240 °C, respectively. The flow rate of nitrogen gas, the carrier gas, was 30 mL min⁻¹. ETH production was expressed as µL h⁻¹ kg⁻¹FW.

Protein extraction

The trichloroacetic acid (TCA)/acetone method was used to extract the total protein (Damerval et al., 1986). Approximately 2.0 g of the sample was manually ground to a fine powder in liquid nitrogen with 0.04 g crosslinking polyvingypyrrolidone (PVPP) and split into 6 tubes. The powder was suspended in 2 mL of acetone containing 10% w/v TCA and 0. 07% v/v β-mercaptoethanol (β-ME). After precipitation at −20 ° C for at least 8 h or overnight, the pellet was collected by centrifuging at 12,000× g for 30 min. Then the powder was suspended in two mL of ice cold acetone (−20 °C) containing 0. 07% (v/v) β-ME at 4 °C for 1 h, then centrifuged at 12,000× g for 20 min, and washed two times. The powder was then suspended in two mL of 80% acetone containing 0. 07% (v/v) β-ME at 4 °C for 30 min, centrifuged at 12,000× g for 15 min, and washed two times. The supernatant was removed and the pellets were dried in a freezer dryer. After air-drying, the powder was dissolved in a hydration solution [7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS), 1% (w/v) DL-Dithiothreitol (DTT)] at a normal temperature for 2 h, then centrifuged at 12,000×g for 30 min. The supernatant was the total protein. The protein content was determined calorimetrically using bovine serum albumin as a standard. Protein samples were stored at −80 °C for further analysis.

2-DE and staining

Sample aliquots containing protein were applied to 17 cm pH 4–7 immobilized pH gradient (IPG) strips, and small volumes of lysis buffer were added to the sample aliquots. Isoelectric focusing was performed on a PROTEAN IEF Cell system (Bio-Rad) for a total of 76 kVh at 20 °C. The voltage was set at 50 V for 14 h, 250 V for 1. 5 h, 1000 V for 2. 5 h, 9000 V for 5 h, and were run at 90000 V and then 500 V for a maximum of 24 h. Next, the gels were run using the PROTEAN II xi cell system (Bio-Rad) at 100 V for 1 h, followed by 250 V until the bromophenol blue nearly reached the bottom of the gel. After electrophoresis, staining was performed by placing the gels into Coomassie brilliant blue solution for 17 h. To provide support for the cucumber proteomics, we performed system optimization of the pH IPG strips, loading quantity and separation gel concentration. The protein spots on the pH 3–10 gels show that the acidic side (pH 3–5) in the region of the protein particle distribution was less than alkaline side (pH 9–10) area, forming a longitudinal tail (Fig. S1). Most of the explant protein spots were focused in the pH 4–7 region. The results showed that 800 µg of loading quantity was optimal (Fig. S2), and 12% (w/v) gel concentration separation was the best choice to obtain the good resolving effect (Fig. S3). The results also showed that pH 4–7 IPG strips (17 cm, nonlinear; Bio-Rad), 800 µg loading quantity and 12% (w/v) separation gel concentration was the most suitable combination for isoelectric focusing of the explants’ total protein in cucumber.

Image analysis and protein identification

The proteins in the gels were visualized by CBB-G250 staining. The gel images were obtained at a resolution of 300 dpi and then imported into PDQuest 8. 0 (Bio-Rad). The protein spots were excised from CBB-stained preparative polyacrylamide gels. The MS and MS/MS data for protein identification were obtained with a MALDI-TOFTOF instrument (Zhang, Zhang & Zhou, 2015). The database NCBI (https://www.ncbi.nlm.nih.gov/) were used to match and identified the protein spots. According to Gene Ontology and UniProt Protein Knowledgebase (http://www.uniprot.org/), the gene ontology (GO) analysis was performed on the proteins identified by mass spectrometry. The proteomic data have been uploaded to the Supplemental Files.

Total RNA extraction and real time PCR

Total RNA from each sample was isolated using a MiniBEST Plant RNA Extraction Kit (TaKaRa, Beijing, China). Total RNA was reverse-transcribed by using the PrimeScript™RT Master Mix (Perfect Real Time) according to the manufacturer’s manual (Takara, Beijing, China). Quantitative real-time PCR was performed with a LightCycler 96 Real- Time PCR System (Roche, Switzerland) using SYBR Premix Ex Taq™ II (TaKaRa, Beijing, China). Every PCR reaction was performed twice, with three independent replicates. The relative expression level of each gene was acquired using a comparative Ct method followed by internal control normalization. PCR was carried out in 20 µL volumes using the following amplification protocol: 10 s at 95 °C, followed by 40 cycles of 15 s at 95 °C, and then annealing at 60 °C for 30 s. The relative quantization of gene expression was calculated and normalized to Actin. The expression change was calculated using the 2^−ΔΔct method. The primers used for PCR analysis are listed in Table 1.

Effects of different concentrations of HRW and ethephon on adventitious root development

To understand the effect of H₂on adventitious root development, cucumber explants were treated with different concentrations of HRW (0, 1%, 10%, 50% and 100%). As shown in Fig. 1, different concentrations of HRW affected the development of adventitious roots. The cucumber explants treated with 50% and 100% HRW produced more adventitious roots than the control explants, but the root number under the 1% HRW treatment was significantly lower than that of the control. The maximum root number was observed with 50% HRW treatment (Fig. 1) and this was used for further study.

Different concentrations of ethephon, ETH-releasing compound, also had significant effects on the development of adventitious roots (Fig. 2). Lower concentrations of ethephon (0. 1, 0. 5, 1, and 10 µmol L⁻¹) increased adventitious root number, while the high concentration (50 µmol  L⁻¹) significantly inhibited adventitious roots. The maximum inducible response was observed in 0. 5 µmol  L⁻¹ ethephon-treated explants (Fig. 2). Thus, we used 0. 5 µmol  L⁻¹ to further investigate the role of ethylene in rooting.

Effects of ethylene inhibitors AVG and AgNO₃ on H₂-induced adventitious root development

To further study the function of ETH in adventitious root formation, ethylene inhibitors were used. Results shown in Figs. S4 and S5 indicate that various concentrations of ETH synthesis inhibitors AVG and AgNO₃ have negative effects on rooting. We used 1 µmol L⁻¹ AVG and 0. 1 µmol L⁻¹AgNO₃ for further study. As seen in Fig. 3, explants treated with either HRW or ethephon alone showed improved adventitious rooting when compared with the control. Although explants treated with HRW plus ethephon had higher root number, they were not significantly higher than explants treated with either HRW or ethephon alone. When AVG (inhibitor of ETH synthesis) or AgNO₃ (an ETH action inhibitor) was added to the HRW, the inducing effects of ethephon on adventitious rooting were reversed (Fig. 3). Thus, we deduce that ETH might be involved in H₂-induced adventitious root development.

Effect of H₂ on ETH production and ETH-related genes’ expression

As shown in Fig. 4A, the production of ETH in 50% HRW treatment was about double that of the control. Moreover, the results in Fig. 4B indicate that HRW treatment significantly enhances the gene expression of CsACS3. When compared with the control, the expression of CsACO1 was noticeably improved by HRW. These consistent results suggested that ETH might function in the downstream of H₂-induced adventitious root formation.

Comparison of 2-DE gels and protein identification during adventitious root development

All 70 protein spots from the 2-DE gels showed statistical differences. However, due to limited genomic information about C. sativus in the NCBI database, only 48 differentially expressed protein spots could be identified by MALDI TOF/TOF MS/MS with a significant search score, shown in Table 2 and Fig. 5. Out of the 48 differential proteins selected by analysis, 15 proteins were down-regulated while 9 (spot 203, 230, 858, 158, 975, 1116, 1132, 626 and 1140) were up-regulated in the HRW treatment; 10 proteins were down-regulated and 4 (spot 2129, 1908, 1561, 1284) were up-regulated in the ethephon treatment; 9 proteins were down-regulated whiles 1 (spot 239) was up-regulated in the HRW + AVG treatment. Table 2 shows a list of the putative names of proteins, matching species, protein score, accession number, molecular weights (MW), isoelectric point (PI), peptides count and Up/Down regulation. The results indicate that matching species listed above originated from the cucumber database, with molecular weights ranging from 9.4 to 75.3 KDa, PI of spots ranging from 4.31 to 8.6, protein scores ranging from 62 to 905, and matched peptide counts between 2 and 27.

Functional analysis of the six identified proteins

Forty-eight protein spots were identified using the annotated NCBI database, and these were designated as 41 known proteins and seven 7 unknown proteins. Using the Gene Ontology and UniProt Protein Knowledgebase, all of the identified proteins were categorized into eight 8 major groups (Fig. 6). Among these, Seventeen photosynthesis-related proteins constituted the largest percentage (35.4%), followed by six energy metabolism-related proteins (12.5%), six translation and transcription-related proteins (12.5%), six protein folding, modification and degradation-related proteins (12.5%), three stress response-related proteins (6.3%), two amino and metabolism-related proteins (4.2%), one cellular cytoskeleton-related proteins (2.1%), and seven unknown proteins (14.6%).

The differentially expressed proteins were identified using mass spectrometry and GO ontology analysis. These proteins were distributed in 3 categories: “biological process” (the largest group), “cellular component” and “molecular function” (the smallest group). As shown in Fig. 7 4 categories of proteins were identified in the biological process, comparing cellular process, metabolic process, response to stimulus and single-organism process; 3 categories of proteins were identified in molecular function, comparing: catalytic activity, transporter activity and binding; and 6 categories of proteins were identified in the cellular components, including organelles, cell parts and so on.

Out of the 41 proteins with known functions, 5 proteins were up-regulated in HRW or ethephon treatment, and were down-regulated in HRW + AVG treatment. These included ribulose-1,5-bisphosphate carboxylase small subunit (Rubisco), sedoheptulose-1,7-bisphosphatase (SBPase), threonine dehydratase (TDH), cytosolic ascorbate peroxidase (CAPX), and protein disulfide-isomerase (PDI) (Table 3). The oxygen-evolving enhancer protein (OEE1) was down-regulated under HRW or ethephon treatment and up-regulated under HRW + AVG treatment.

Validation of the identified proteins at the mRNA level during adventitious root development

The mRNA levels of Rubisco, SBPase, TDH, CAPX and PDI genes under HRW or ethephon treatment were significantly higher than those of the control (Fig. 8). However, HRW+AVG treatment caused significant reduction in the expression of these genes. The mRNA levels of OEE1 increased significantly in the explants treated with HRW or ethephon, and decreased significantly in the explants treated with HRW+AVG. The changes of the mRNA levels of 6 genes were consistent with protein levels observed in the 2-DE results.