Hydrogen Peroxide Is a Second Messenger in the Salicylic Acid-Triggered Adventitious Rooting Process in Mung Bean Seedlings

In plants, salicylic acid (SA) is a signaling molecule that regulates disease resistance responses, such as systemic acquired resistance (SAR) and hypertensive response (HR). SA has been implicated as participating in various biotic and abiotic stresses. This study was conducted to investigate the role of SA in adventitious root formation (ARF) in mung bean (Phaseolus radiatus L) hypocotyl cuttings. We observed that hypocotyl treatment with SA could significantly promote the adventitious root formation, and its effects were dose and time dependent. Explants treated with SA displayed a 130% increase in adventitious root number compared with control seedlings. The role of SA in mung bean hypocotyl ARF as well as its interaction with hydrogen peroxide (H2O2) were also elucidated. Pretreatment of mung bean explants with N, N’-dimethylthiourea (DMTU), a scavenger for H2O2, resulted in a significant reduction of SA-induced ARF. Diphenyleneiodonium (DPI), a specific inhibitor of membrane-linked NADPH oxidase, also inhibited the effect of adventitious rooting triggered by SA treatment. The determination of the endogenous H2O2 level indicated that the seedlings treated with SA could induce H2O2 accumulation compared with the control treatment. Our results revealed a distinctive role of SA in the promotion of adventitious rooting via the process of H2O2 accumulation. This conclusion was further supported by antioxidant enzyme activity assays. Based on these results, we conclude that the accumulation of free H2O2 might be a downstream event in response to SA-triggered adventitious root formation in mung bean seedlings.


Introduction
Roots function as the interface between plants and the terrestrial environment. In higher plants, the root system is composed of primary roots, lateral roots and adventitious roots. Primary roots are initiated during embryogenesis and elongate after germination. Compared with primary roots, lateral roots and adventitious roots are 'post-embryonic' roots, as they initiate from non-pericycle tissues. Lateral roots initiate from primary roots or axes, and adventitious roots initiate from stem and leaf-derived cells. Together, these plant roots form the root system, the architecture of which can be altered in response to environmental changes and stimuli [1].
The appearance of adventitious roots may date the evolution of endogenous initiation combined with reverse auxin transport because these roots appear to have evolved repeatedly over time, and it is suggested they may have been required for the establishment of vascular continuity [2]. Adventitious root formation (ARF) is part of the normal development of plants and occurs naturally. ARF is useful for facilitating the uptake of water and nutrients from the soil, the anchorage of plants to substrates and the formation of food storage reserves.
The plant hormone SA is an endogenous growth regulator with a phenolic nature that plays a critical role in many diverse physiological processes. A well-known effect of SA is increasing the temperature in thermogenic plants [25] and flowering [26,27]. Previous studies also support the notion that SA is involved in modulating the plant response to many abiotic and biotic stresses, such as disease resistance [28,29] photosynthesis [30,31], low temperature resistance [32,33], drought resistance [34,35], salt resistance [36,37] and fruit maturity [38], among others. SA is also known to play a possible role in activating the defense response of plants to pathogen attack. SA mediates the oxidative burst that leads to programmed cell death (PCD) in the hypersensitive response, and it has been suggested that SA could act as a signal in the development of the SAR [39,40].
Nevertheless, defining the biological functions of SA in the context of the adventitious rooting process has been controversial. Still and Kling advocated that supplying SA could slight stimulate adventitious root initiation [41,42]. And in combination with indoleacetic acid (IAA), SA act synergistically effect [42]. This study revealed a distinctive role for SA in IAAinduced adventitious root formation. In 1989, Riov found that SA could effectively stimulate rooting of mung bean cuttings. However, only two concentration of SA has been examined (0.1 mM, 0.2 mM) [5]. Using SA deficient mutants, Gutierrez suggested that SA is possibly a positive regulator of adventitious rooting in Arabidopsis [43]. On the other side, Kang found that SA could increase the amount of scopolamine in adventitious root cultures of Scopolia parviflora, and without any negative effects on growth [44]. In contrast, another study yielded completely different results. Li reported that SA inhibited adventitious root formation and decreased the weights of roots in mung bean hypocotyls cuttings [45]. And De Klerk indicated that SA inhibited IAA-induced adventitious root formation in apple microcuttings during the initial posttreatment days. These findings may be the result of SAenhanced IAA decarboxylation [46]. To date, the confirmatory role of SA in ARF is still ambiguous.
The main object of this study was to identify the role of SA in ARF in mung bean hypocotyls. Through pharmacological and physiological approaches, we demonstrate that SA is an important factor that induces adventitious root organogenesis and formation in mung bean hypocotyls. We also examine the interaction between SA with another important second signaling molecule, H 2 O 2 . Furthermore, we provide evidence that SA can elevate H 2 O 2 levels through the regulation of the antioxidant enzymes in mung bean seedlings. Based on these results, a simple transduction pathway model is proposed, wherein H 2 O 2 acts as a downstream messenger in the SAmediated signaling pathway that induces ARF.

Plant Material
Mung bean (Phaseolus radiatus L) seeds were washed in distilled water and immersed in 0.1% HgCl 2 for 5 minutes. After five washes in distilled water, the seeds were soaked in distilled water for 12 h at 27°C and were then maintained at 27°C for 5 d, with a 14-h photoperiod (PAR of 200 µmol m −2 s −1 ).

Explant Treatments
The explants consisted of a terminal bud, two primary leaves and 4 cm of the hypocotyls. After the primary roots were removed, the explants were put into 100-ml beakers containing 50 ml of distilled water (control) or 50 ml of the test solution for 24 h under the same conditions. After being washed three times, the seedlings were moved into distilled water, used as explants and maintained under the same temperature and photoperiod conditions for another five days. The distilled water was replaced daily.

Application of plant hormones and inhibitors
All chemicals were purchased from Sigma-Aldrich (St Louis, USA). SA was dissolved in ethanol for a final stock concentration of 1 M. The H 2 O 2 scavenger DMTU was dissolved in distilled water to make a stock solution of 10 M. The NADPH oxidase inhibitor DPI was dissolved in dimethylsulfoxide (DMSO) to make a stock solution of 1 mM. For the DMTU and DPI treatments, after the removal of the primary roots, mung bean seedlings were pretreated in beakers for 4 h in the presence of DMTU or DPI and were then moved into test solutions for another 24 h. In addition, all of the explants treated with the inhibitors tested in this study appeared healthy.

Statistical analyses
The numbers of adventitious roots were determined after five days of treatment. The number of adventitious roots of more than 1 mm long was recorded. The data presented are the means of at least three independent experiments with 30 explants per treatment. The data were analyzed using an ANOVA. The comparisons between the mean values were conducted using the least significant difference (LSD) test, with the significance set at P < 0.05, and the standard error (S.E.) was calculated. The statistical analyses were performed using SPSS software version 14.0 (SPSS Inc., Chicago, IL).

Root primordium observation
Root primordium observation was accomplished using Feulgen-staining, as described by Kordan [47]. After being treated for 48 h, the hypocotyls cut from the explants were fixed in absolute ethanol:glacial acetic acid (3:1) for 48 h and were then stored in 70% ethanol until they were required for use. The hypocotyls were rehydrated in an ethanol series (70-50-30-10%), hydrolyzed for 10 min in 10% HCl at 60°C and then placed in Schiff's reagent (24 h) followed by a thorough washing with tap water until the wash water was no longer pink. The Feulgen-stained hypocotyls were placed in a 10% aqueous glycerine solution in uncovered plastic Petri dishes, and the glycerine allowed for concentration via the gradual evaporation of the water on a warming plate at 40°C. The stained hypocotyls were examined as whole mounts in the concentrated glycerine in Petri dishes using bright field transmission optical microscopy.

Visualization of H 2 O 2 with the DAB method
H 2 O 2 was detected with the DAB method [48]. Briefly, the hypocotyls cut from the explants were treated with 1 mg/ml solution of DAB, pH 3.8, for 8 h under light at 25°C. The treated hypocotyls were then placed in boiling 95% ethanol for 10 min to decolorize the hypocotyls, except for the deep brown polymerization product produced by the reaction of DAB with H 2 O 2 . After cooling, the hypocotyls were extracted with fresh ethanol and preserved at 4°C in ethanol and photographed.

Cytochemical detection of H 2 O 2
H 2 O 2 was visualized at the subcellular level using CeCl 3 for localization [49]. Electron-dense CeCl 3 deposits are formed in presence of H 2 O 2 and are visible via transmission electron microcopy. Tissue samples (2×2 mm 2 ) were excised from the hypocotyls of the cuttings and then vacuum infiltrated with freshly prepared 5 mM CeCl 3 in 50 mM 3-(N-morpholino)propane-sulfonic acid at pH 7.2 for 30 min. The tissue samples were then fixed in 1.25% (v/v) glutaraldehyde and 1.25% (v/v) paraformaldehyde in 50 mM sodium cacodylate buffer (CAB), pH 7.2, for 1 h at room temperature and kept overnight at 4°C. After fixation, the tissue samples were washed twice for 10 min in CAB and postfixed for 45 min in 1% (v/v) osmium tetroxide in CAB. The tissue samples were then washed twice for 10 min in CAB and dehydrated in a graded acetone series (30,50,70,80,90 and 100% [v/v]), progressively embedded in rising concentrations of acetone-resin mixtures, and incubated over two 24 h replacements of pure epoxy resin before polymerization at 60°C for 48 h. Ultrathin sections (50 to 100 nm) were obtained with a Reichert Ultracut E Microtome, using a diamond knife mounted on nickel grids (200 mesh), and examined without staining with a transmission electron microscope (H7650, HITACHI, Japan) at an accelerating voltage of 80 kV.

Endogenous H 2 O 2 assay
H 2 O 2 content was measured by monitoring the A 415 of the titanium-peroxide complex, following the method described by Brennan and Frenkel [50], with slight modifications. Briefly, 0.5 g (FW) of hypocotyl material was frozen in liquid nitrogen immediately after completion of the treatment period and ground with liquid nitrogen, and the fine powdered material was mixed with 5 ml of cooled acetone in an ice bath. The mixture was centrifuged at 10,000 g for 15 min (4°C). Next, 1 ml of supernatant was obtained, and this was followed by the addition of 0.2 ml of a titanium reagent (20% w/v) and 0.4 ml of an ammonium solution to precipitate the titaniumhydroperoxide complex. The reaction mixture was centrifuged at 10,000 g for 10 min. The precipitate was dissolved in 5 ml of 2 M H 2 SO 4 . The supernatant's absorbance was measured at 415 nm against blanks using a LabTech UV1600 spectrophotometer (LabTech Inc. USA). A standard response curve was prepared with known concentrations of H 2 O 2 using the same method as described above. The H 2 O 2 content was calculated through comparison with a standard graph generated with known H 2 O 2 concentrations.

Determination of hypocotyl O 2 -production
O 2 -production was measured, as described by Able [51], by monitoring the reduction of XTT in the presence of O 2 -. Mung bean hypocotyls (1 g) were homogenized with 5 ml of 50 mM Tris-HCl buffer (pH 7.5) and centrifuged at 15,000 g for 10 min. The reaction mixture (1 ml) contained 50 mM Tris-HCl buffer (pH 7.5), 50 μg of plasma membrane proteins, and 0.5 mM XTT. The reduction of XTT was evaluated at 470 nm for 5 min. Corrections were made for the background absorbance in the presence of 50 units of SOD. The O 2 -production rate was calculated using an extinction coefficient of 2.16×10 4 M -1 cm -1 .

Isolation of the plasma membrane and determination of NADPH oxidase activity in the plasma membrane
Plasma membrane vesicles were isolated from mung bean hypocotyls using two-phase partitioning, according to a procedure described previously [52]. The membrane vesicles were resuspended in a 50 mM Tris-HCl buffer, pH 7.5 and used immediately for NADPH oxidase activity assays. The membrane vesicles (10 μg) were incubated in the reaction buffer (50 mM Tris-HCl buffer, pH 7.5, and 0.5 mM XTT). The reaction was initiated with the addition of NADPH. After 10 min at 25°C, the reaction solution was used for the spectrophotometric analysis of XTT formazan production at A 470 . The NADPH activity was expressed as △A 470 per milligram of protein per minute. △A 470 represents the difference in XTT formazan absorbance at 470 nm in the presence and absence of 100 units of SOD.

Enzyme activity assays
For the determination of enzyme activity, plant material was frozen in liquid N 2 and stored at -80°C until the plants were ground and enzymes were extracted. Frozen root segments (0.3 g) were homogenized in 5 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1% polyvinylpyrrolidone, with the addition of 1 mM ASC in the case of the APX assay. The homogenate was centrifuged at 15,000 g for 20 min at 4°C, and the supernatant was immediately used for the following described enzyme assays. Protein content was determined, according to the Bradford method, with BSA as a standard. SOD activity was assayed by monitoring the inhibition of the photochemical reduction of nitro blue tetrazolium (NBT), according to the method of Giannopolitis and Ries [53]. One unit of SOD activity was defined as the amount of enzyme that was required to produce 50% inhibition of the reduction of nitro blue tetrazolium as monitored at 560 nm.
CAT activity was assayed by measuring the rate of decomposition of H 2 O 2 at 240 nm, as described by Aebi [54].
GR activity was determined, as described by Connell and Mullet [55]. The activity of the enzyme was assessed in NADPH-containing HEPES buffer by monitoring the decrease in absorbance at 340 nm as NADPH is oxidized.
APX activity was determined by following the decrease in 290 nm for 3 min in a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ASC, 0.1 mM H 2 O 2 , and 200µl of enzyme extract. The reaction was started by the addition of the enzyme extract. The reaction was corrected for the low non-enzymatic oxidation of ASC by H 2 O 2 [56].

SA induces adventitious rooting in mung bean hypocotyls in a dose-and time-dependent manner
To investigate the effect of SA on ARF in mung bean hypocotyls, 5-d-old seedlings with their primary roots removed were used as explants and incubated in a control treatment (water) and different concentrations of SA (0.1, 0.2, 0.4, 0.6 and 0.8 mM) at the same temperature and with the same photoperiod conditions for 24 h. After 5 days, the root number was analyzed and quantified. The results indicated that exogenous SA could promote adventitious root formation, and its effects were dose and time dependent (Figure 1). The treatment of the seedling explants with a low concentration of SA (0.2 mM) slightly promoted ARF, and the root number of treatment explants exposed to 0.4 mM SA was 2.3-fold compared with the explants in the control treatment. An inhibitory effect on ARF was observed when the SA concentration was 0.8 mM. These results indicate that exogenous SA at particular concentrations promotes adventitious root formation in mung bean hypocotyl cuttings. Therefore, an SA concentration of 0.4 mM was used in the further experiments.

Effect of SA on root primordia formation
An anatomical study was performed to observe the primordium formation during the first stages of adventitious rooting. We analyzed (0-1 cm) sections of mung bean hypocotyls treated with water (control) or SA (0.4 mM SA). A photograph of a representative root primordium observation is presented in Figure 2b. At 48 h after the removal of the primary root system, adventitious root primordium formation was detected in explants treated with SA and water. In the watertreated plants, few root primordia could be observed, whereas in the SA-treated explants, we observed a greater number of root primordia than in the water-treated hypocotyls. The number of root primordia in the SA-treated explants was more than 2-fold higher than in the control seedlings. These results suggested that SA promotes AR formation in mung bean hypocotyls, possibly due to the induction of the dedifferentiation

Interaction between SA and H 2 O 2 in ARF in mung bean hypocotyls
The above data indicate a SA-dependent effect on the formation of adventitious roots in mung bean hypocotyls. Previous work has shown that H 2 O 2 can stimulate ARF in mung bean hypocotyls, and H 2 O 2 might act as a downstream signaling molecule in IAA-induced ARF [57][58][59] (Figure 3).

DMTU and DPI prevent SA-induced adventitious root formation
To further elucidate the role of H 2 O 2 in SA-triggered ARF in mung bean hypocotyls, the role of N, N'-dimethylthiourea (DMTU; a cell-permeable scavenger for H 2 O 2 ) [60][61][62][63][64] in mung bean hypocotyls was investigated. We determined that H 2 O 2 depletion treatment provoked a 2-fold reduction in the root number in comparison with the control explants. We believe that the inhibitory effect of DMTU on ARF can mainly be attributed to the scavenging of H 2 O 2 . However, pretreating hypocotyls with DMTU for 4 h, followed by a transfer into 0.4 mM SA for another 24 h, result indicated that DMTU can partially ameliorate the effect of SA on ARF (Figure 4a). The inhibition of the promotional effect of SA on ARF in mung bean hypocotyls that were pretreated with DMTU indicates that H 2 O 2 may play a pivotal role in the SA-induced adventitious rooting process.
The above experiments identified that endogenous H 2 O 2 might be involved in SA-induced ARF. The production of intracellular H 2 O 2 was mainly attributable to NADPH oxidase, which converts O 2 to superoxide anions (O 2 -) and then to H 2 O 2 [65][66][67]. Diphenyleneiodonium (DPI) is a specific inhibitor of membrane-linked NADPH oxidase that consequently inhibits  [60,[68][69][70][71]. The effect of DPI treatment on ARF in mung bean hypocotyls was also investigated. As shown in Figure 4b, pretreatment with DPI alone could depress ARF. We also found that pretreatment with DPI could significantly depress the SA induction of ARF. These results imply that H 2 O 2 formation might be required for SA-induced ARF. The observed changes in the H 2 O 2 content after SA treatment further support this conclusion.

SA enhances H 2 O 2 levels in mung bean hypocotyls
To further confirm the interaction between SA and H 2 O 2 on ARF in mung bean hypocotyl cuttings, we measured the time course of endogenous H 2 O 2 levels in the hypocotyls of mung beans incubated with water and SA. In this study, SA treatments enhanced H 2 O 2 levels in the hypocotyls of mung bean seedlings in a time-dependent manner. The treatment of hypocotyls with 0.4 mM SA significantly enhanced in vivo H 2 O 2 contents compared with hypocotyls treated with water. Here, a rapid increase in the endogenous H 2 O 2 level was detected during the first 12 h of hypocotyl incubation with SA, with a peak reached at 12 h; however, there was only a slight increase in the H 2 O 2 level in the water-treated hypocotyls (Figure 5a). The H 2 O 2 content at 3 h and 6 h was increased by 48% and 61%, respectively, compared with the control seedlings. After 12 h of treatment, the production of H 2 O 2 reached maximum levels and increased by 33% compared with the control value. The results obtained in this study confirm that SA-mediated ARF has a close association with H 2 O 2 accumulation. However, the SA treatment did not affect the production of O 2 -throughout the treatment period (Figure 5b). In this study, diaminobenzidine (DAB) uptake method has been adapted of in-situ detection of H 2 O 2 [48]. The development of the DAB-H 2 O 2 reaction product in hypocotyls in response to cutting was shown in Figure 6a. The color was more intense in SA incubated cuttings than water treated seeding, so did the stained area. Histochemical and cytochemical have been used for the detection of H 2 O 2 generated in mung bean hypocotyls. H 2 O 2 was visualized at the subcellular level using CeCl 3 for localization [49]. The greatest accumulation of H 2 O 2 was observed in the cell wall and intercellular space in SA treated cutting after 12 h incubation, however, there is barely deposits of CeCl 3 in water-treated seedling (Figure 6b).

Influence of SA on NADPH oxidase activity and antioxidant enzymes
NADPH oxidase is a protein that transfers electrons from NADPH to an electron acceptor, which leads to the formation of reactive oxygen species. To study a possible link between SA and NADPH oxidase, the activity of NADPH oxidase was measured. Plasma membrane (PM) vesicles were isolated from roots, and the NADPH oxidase activity was determined by measuring O 2 -production. The results indicate that SA treatment did not affect NADPH activity (date not shown) Altered ROS levels can result either from increased production or decreased scavenging. The activities of the antioxidant enzymes in the cuttings treated with water or SA were investigated to determine the source of H 2 O 2 formation. As shown in Figure 7a, a significant enhancement in the activity of SOD occurred within the first 6 h of incubation with SA. The SA-induced SOD activity was enhanced by 105% compared with the control value after 3 h of incubation. After 6 h of incubation, the activity of SOD was increased by 69% compared with the control seedlings. It can be presumed that the accumulation of H 2 O 2 observed with the SA treatment could be a result of SOD activity enhancement. Accordingly, the pretreatment with SA significantly repressed the activities of CAT in a time-dependent manner (Figure 7b). The CAT activities at 3 h and 6 h were decreased by 12% and 15%, respectively, compared with the control seedlings. After 12 h of treatment, the CAT activity was decreased by 17% compared with the control value. However, there was no significant change in APX and GR activities between the water and SA treatments (Figure 7c, 7d).

Discussion
AR at the base of plant cuttings is an innate de novo organogenesis process that allows the massive vegetative propagation of many economically and ecologically important species [72]. It is necessary to understand the physiological and biochemical process of adventitious rooting. The discovery of signal molecules involved in the intricate network that triggers ARF remains a major goal for a large number of biotechnological procedures. Although a variety of plant components induce ARF and signal transduction has been identified, the molecular mechanism underlying meristem initiation is still undefined and remains to be proven.
SA is believed to be a key signaling molecular in SAR against pathogens and to play an important role in mediating plant responses to a variety of abiotic stresses. However, the effects of SA in root development are less known. A previous study demonstrated that SA might participate in root formation and development in plants. Some researchers observed that an aqueous solution of SA sprayed on the shoots of soybeans could significantly increase the growth of shoots and roots. In addition, SA induces increases in root growth of up to 100% in the field [73]. Singh [74] observed that SA stimulated root formation in the young shoots of ornamental plants. Salicylic acid and its derivatives are more closely related to these structural requirements. In faba bean (vicia faba L.), SA its chemical derivative (acetylsalicylic acid, ASA), at appropriate concentrations could increased rooting efficiency [75]. In Pb 2+ stress seedlings, SA pretreatment also could significantly increase the length of shoots and roots and partially protect seedlings from Pb 2+ toxicity [76]. Some researchers have observed that SA promotes later root initiation, emergence and growth, possibly via crosstalk with cytokinin or auxin [77]. However, only a few researchers have noted the role of SA in ARF and the intricate network involving SA and other molecules in this procedure. Our study indicates that SA alone is directly involved in the adventitious rooting process in mung bean hypocotyl cuttings. A dramatic increase of the AR number was observed in SA-treated mung bean hypocotyls cuttings compared with control seedlings (Figure 1a, Figure 2a). The treatments performed with different concentrations of SA confirmed that the effect was dose dependent, with an optimum concentration at 0.4 mM. Those results are consistent with the report of Kling and Riov, who advocated that application of SA increased AR number and yield in mung bean (Vigna rediata L.) [5,42]. In addition, SA induced adventitious rooting in a time-dependent manner (Figure 1b). Furthermore, we observed that SA promotes this process through the differentiation of cells to reestablish a new apical meristem (Figure 2b). Accordingly, we confirmed that SA is involved in the adventitious rooting process in mung bean hypocotyl cuttings. H 2 O 2 is a diffusible multifunctional molecule involved in numerous physiological processes in phylogenetically distant species. It is usually regarded as a cytotoxic molecule in cell metabolism. However, there is strong evidence that indicates that H 2 O 2 is a useful second messenger in plant growth and development and that it mediates various physiological and biochemical processes, including SAR [78,79], stomata closure [80,81], programmed cell death (PCD) [82,83], wounding signaling [49], and root gravitropism [84]. Other researchers have suggested that H 2 O 2 might possibly be an integral component of intracellular signaling transduction. Guan indicated that H 2 O 2 plays an important intermediary role in the ABA signal transduction pathway, which leads to the induction of the Cat1 gene [85]. Pei noted that Ca 2+ -channel activation by H 2 O 2 is an important mechanism for ABA-induced stomatal closing [71]. H 2 O 2 also regulates plant cell expansion through the activation of Ca 2+ channels via a NADPH pathway [86]. In addition, there is also a considerable cross talk between H 2 O 2 and NO transduction [87].
Increasing evidence supports the notion that H 2 O 2 plays a causal role in lateral root development and adventitious root formation. Su and others [64] observed that H 2 O 2 generated by polyamine oxidative degradation is involved in the development of later roots in soybean plants. Furthermore, Li reported that exogenous H 2 O 2 is an essential component in ARF in mung bean and cucumber hypocotyls cuttings and acts as a second massager in IAA-induced ARF [57,58], yet the mechanisms of H 2 O 2 in ARF remain elusive.
Early evidence indicates that SA and H 2 O 2 are involved in the responses of plants to pathogens [88][89][90]. Although many works have studied the specific interaction between SA and H 2 O 2 in plants, whether SA and H 2 O 2 participate in ARF remains unclear. In this study, we observed that SA and H 2 O 2 played a synergetic role in regulating ARF in mung bean hypocotyl cuttings (Figure 3). The treatment of hypocotyls with SA (SA 0.4 mM) plus H 2 O 2 (10 mM) resulted in an increased response and an increased root number in comparison with hypocotyls treated with SA or H 2 O 2 alone. Interestingly, a high level SA and H 2 O 2 led to a significant suppress in the number of AR. This might be the exacerbation of oxidative stress, resulting in the inhibitory effect on ARF. Accordingly, we can speculate that SA and H 2 O 2 have additive effect in adventitious rooting process. These results indicate that there is a potential interaction between SA and H 2 O 2 as signaling molecules.
However, it is still unclear whether H 2 O 2 involves signal transduction in relation to the development of SA-induced adventitious rooting. The development of adventitious roots are blocked in hypocotyls treated with DMTU or DPI (Figure 4), implying that endogenous H 2 O 2 plays a pivotal role in adventitious rooting. These results suggest that DMTU and DPI achieve inhibition via a decrease in the H 2 O 2 level during the adventitious rooting process. However, when DMTU was used as a trap for H 2 O 2 , the number of SA-induced ARF was significantly reduced, suggesting that SA requires H 2 O 2 to initiate adventitious rooting. DPI, a specific inhibitor of membrane-linked NADPH oxidase, which is one of the main sources of H 2 O 2 formation in plant cells, inhibited NADPH oxidase activity and also inhibited ARF in mung bean hypocotyls. Furthermore, the application of a specific concentration of SA alleviated the inhibitory effect of DPI (Figure 4b). The results suggest that H 2 O 2 may function as a downstream signaling molecule or involved in a parallel pathway in the SA-induced formation and development of AR in mung bean seedlings.
In recent years, considerable evidence has accumulated suggesting that physiologically relevant concentrations of SA treatment can enhance H 2 O 2 levels, and H 2 O 2 has been proposed as being functionally downstream of SA in plants based on the evidence that SA can participate in regulating the antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) [61,78,[91][92][93][94].. In adventitious rooting process, Tewari reported that SA could induce the hydrogen peroxide accumulation in Panax ginseng [95]. Furthermore, 40 day-old adventitious roots of Panax ginseng treated with 0.2 mM SA could cause an increase in the carbonyl and hydrogen peroxide contents [76]. In contrast, other studies report that SA accumulation could be induced by elevated H 2 O 2 levels [97,98]. In addition, H 2 O 2 activates SA biosynthesis via stimulation of BA2H (benzoic acid 2-hydroxylase) activity [97], which is also observed in tobacco cells. Furthermore, other studies have indicated that H 2 O 2 does not function downstream of SA in regulating PR protein expression [99]. In view of the existing contradictory results derived from in vitro and in vivo studies, the method by which SA enhances H 2 O 2 production in vivo and the relative significance of such SA-enhanced H 2 O 2 remains unclear.
However, there is no information, as far as we know, regarding the effects of SA on H 2 O 2 generated in the adventitious rooting process of mung bean. The interaction between exogenous SA and H 2 O 2 on ARF in mung bean hypocotyl cuttings was further confirmed by the vivo H 2 O 2 content determination. The present findings indicate that the enhanced endogenous H 2 O 2 level was triggered by SA treatment in a time-dependent manner (Figure 5a). A peak H 2 O 2 content was detected in mung bean hypocotyls incubated with SA for 12 h after the primary root was removed. These results indicate that the rapid increase in H 2 O 2 content during the first 12 h of incubation could be attributed to the SA stimulus. Nag and others [14] demonstrated that in mung bean hypocotyls cuttings, the induction, initiation and expression phases are 0-24, 24-72 and after 72 h, respectively. We believe that the enhancement of H 2 O 2 production by SA treatment is useful for cell division and root primordium in the induction phase. Nevertheless, hypocotyl cuttings treated with SA did not present differences in O 2 -content throughout the entire experimental period compared with the water-treated seedlings ( Figure 5b) [102,103]. It has been reported that SA treatment was most effective in increasing NADPH oxidase activity in wheat seedlings [104]. To address this issue, we focused our attention on the hypothesis that adventitious root formation induction by SA signals may be involved in the NADPH oxidase pathway. In the end, we observed that there was no difference in the NADPH oxidase activity between the water and SA treatments (date not shown). So, we thought that SA-induced H 2 O 2 accumulation had no relationship with the NADPH oxidase signal pathway.
To further investigate the status of the antioxidant defense system in SA-treatment seedlings, the activity of the major ROS-scavenging enzymes, SOD, CAT, APX and glutathione reductase (GR) were investigated. SOD converts highly reactive O 2 -to the more stable molecular-H 2 O 2 . Previous results reported by Rao demonstrated that SA could increase SOD activity in Arabidopsis thaliana, which led to increases in the H 2 O 2 content [61]. In the current paper, higher SOD activity in SA-treated hypocotyls was observed to be time dependent. In addition, the highest SOD activity was observed at 6 h after incubation with SA, and SOD activity increased by 69% compared with the control seedlings ( Figure 7a). The enhanced SOD activity in SA-treated hypocotyls might participate in the formation of H 2 O 2 . The existing relationship between SA and SOD activity clearly suggests that hypocotyls treated with SA may have enhanced H 2 O 2 largely due to the activation of enzymes capable of generating H 2 O 2 . H 2 O 2 can be removed by CAT or by the APX of the ascorbate-glutathione antioxidant cycle [105]. Previous results indicate that SA inhibits CAT activity and thereby causes an increase in H 2 O 2 levels [32,78,106]. In the present study, lower CAT activity was detected in SA-treated hypocotyls compared with the watertreated hypocotyls (Figure 7b). These results are in agreement with the hypothesis that SA enhances H 2 O 2 levels by inactivating the enzymes that are capable of degrading H 2 O 2 . It can be concluded that the accumulation of H 2 O 2 observed in SA-treated hypocotyls also could be the result of the inhibition of CAT activity. High GR activity maintains the pool of glutathione in the reduced state, allowing GSH to be used by DHAR to reduce DHA to AA [107]. Nevertheless, there was no difference in APX and GR activity between the SA and watertreated cuttings (Figure 7c, 7d). Therefore, APX and GR activity were not the cause of H 2 O 2 accumulation in the present study. It can be concluded that the accumulation of H 2 O 2 observed with SA treatment mainly resulted from the enhancement of SOD activity and the inhibition of CAT activity. This mechanism may explain the increase of in vivo H 2 O 2 content at 12 h after SA incubation. Comparisons in Figure 6 indicate that SA-enhanced H 2 O 2 levels were related to the increased activities of SOD and were dependent on CAT changes, but that was not the case with GR activity and APX activity. Thus, the conversion of O 2 -by SOD does appear to be the main reason for altered H 2 O 2 levels.
In conclusion, this study contributes to defining a distinctive role for SA during the adventitious rooting process in mung bean hypocotyls. In addition, hypocotyls treated with SA presented a transient elevation in H 2 O 2 content. Furthermore, the results of this study indicate that SA enhanced H 2 O 2 via changes to the status of the antioxidant system. Based on this evidence, we suggest that H 2 O 2 could be involved in the signaling pathway transduction of SA that triggered ARF in the examined mung bean hypocotyls. However, the underlying mechanism by which SA and H 2 O 2 induced adventitious root formation requires further investigation.