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

Abstract

Previous studies have shown that both hydrogen gas (H₂) and ethylene (ETH) play positive roles in plant adventitious rooting. However, the relationship between H₂and ETH during this process has not been explored and remains insufficiently understood. In this study, cucumber (Cucumis sativus L.) was used to explore the proteomic changes in ETH-H₂-induced rooting. Our results show that hydrogen-rich water (HRW) and ethylene-releasing compound (ethephon) at proper concentrations promote adventitious rooting, with maximal biological responses occurring at 50% HRW or 0.5 µM ethephon. ETH inhibitors aminoethoxyvinylglycine (AVG) and AgNO₃ cause partial inhibition of adventitious rooting induced by H₂, suggesting that ETH might be involved in H₂-induced adventitious rooting. According to two-dimensional electrophoresis (2-DE) and mass spectrometric analyses, compared with the control, 9 proteins were up-regulated while 15 proteins were down-regulated in HRW treatment; four proteins were up-regulated while 10 proteins were down-regulated in ethephon treatment; and one protein was up-regulated while nine proteins were down-regulated in HRW+AVG treatment. Six of these differentially accumulated proteins were further analyzed, including photosynthesis -related proteins (ribulose-1,5-bisphosphate carall boxylase smsubunit (Rubisco), sedoheptulose-1,7-bisphosphatase (SBPase), oxygen-evolving enhancer protein (OEE1)), amino and metabolism-related protein (threonine dehydratase (TDH)), stress response-related protein (cytosolic ascorbate peroxidase (CAPX)), and folding, modification and degradation-related protein (protein disulfide-isomerase (PDI)). Moreover, the results of real-time PCR about the mRNA levels of these genes in various treatments were consistent with the 2-DE results. Therefore, ETH may be the downstream signaling molecule during H₂- induced adventitious rooting and proteins Rubisco, SBPase, OEE1, TDH, CAPX and PDI may play important roles during the process.

Keywords: Adventitious root formation; Cucumber; Ethylene; Hydrogen gas; Proteomics.

Figure 1. Effects of different concentrations of HRW on adventitious root development.

The primary root system was removed from hypocotyls of 7-day-old germinated cucumber. Explants were incubated with distilled water or different concentrations of HRW and indicated for 7 days. The number (A) of adventitious roots per explant were expressed as mean ±  SE (n = 20 explants from each of four independent experiments). Bars with different lowercase letters were significantly different (Duncan’s multiple range test, P < 0.05. Photographs (B–F) show hypocotyl explants after 7 days of the treatments indicated.

Figure 2. Effects of different concentrations of ethephon on adventitious root development.

The primary root system was removed from hypocotyls of 7-day-old germinated cucumber. Explants were incubated with distilled water or different concentrations of ethephon and indicated for 7 days. The number (A) of adventitious roots per explant were expressed as mean ±  SE (n = 20 explants from each of four independent experiments). Bars with different lowercase letters were significantly different (Duncan’s multiple range test, P < 0.05. Photographs (B–G) show hypocotyl explants after 7 days of the treatments indicated.

Figure 3. Effects of ETH inhibitor on H₂-induced adventitious root development.

The primary root system was removed from hypocotyls of 7-day-old germinated cucumber. Explants of cucumber were incubated with 0.5 µM ethephon, 50% HRW, 50% HRW + 0.5 µM ethephon, 50% HRW + 1 µM AVG, 50% HRW+ 0.1 µM AgNO₃/NaNO₃ and indicated for 7 days. The number (A) of adventitious roots per explant were expressed as mean ±  SE (n = 20 explants from each of four independent experiments). Bars with different lowercase letters were significantly different (Duncan’s multiple range test, P < 0.05. Photographs (B–G) show hypocotyl explants after 7 days of the treatments indicated.

Figure 4. The effect of HRW on the ETH production (A) and on the expression level (B) of two ETH-related genes in cucumber explant.

The values (means ± SE) are the average of three independent experiments. Bars with an asterisk presents significant (P < 0.05), and two asterisks presents very significant (P < 0.01).

Figure 5. Separation of the total proteins extracted from the explants of cucumber on 2-DE gels over the pH range 4–7.

Each map depicts one representative gel (of three replicates). A total of 48 protein spots showing difference are numbered on the gels. Arrows indicate the protein spots that were positively identified. (A) Control. (B) HRW. (C) Ethephon. (D) HRW + AVG.

Figure 6. The functional classification and distribution of all 41 identified proteins.

Unknown proteins include those whose functions have not been described.

Figure 7. The gene ontology (GO) analysis of the identified proteins.

Information of number and percentage of involved proteins in a term are shown in left and right y-axis.

Figure 8. The effect of ETH and HRW on the expression levels of rbcS (A), SBPase (B), TDH (C), OEE1 (D), CAPX (E) and PDI (F).

The values (means ± SE) are the average of three independent experiments. Bars with different lowercase letters were significantly different by Duncan’s multiple range test (P < 0.05).