Hydrogen Sulfide Promotes Root Organogenesis in Ipomoea batatas, Salix matsudana and Glycine max

Hua Zhang1∗#, Jun Tang2#, Xiao-Ping Liu3, Yun Wang1, Wei Yu1, Wei-Yan Peng1, Fang Fang1, Dai-Fu Ma2∗, Zhao-Jun Wei1 and Lan-Ying Hu1

Abstract

In this report, we demonstrate that sodium hydrosulfide (NaHS), a hydrogen sulfide (H2S) donor, promoted adventitious root formation mediated by auxin and nitric oxide (NO). Application of the H2S donor to seedling cuttings of sweet potato (Ipomoea batatas L.) promoted the number and length of adventitious roots in a dose-dependent manner. It was also verified that H2S or HS− rather than other sulfur-containing components derived from NaHS could be attributed to the stimulation of adventitious root formation. A rapid increase in endogenous H2S, indole acetic acid (IAA) and NO were sequentially observed in shoot tips of sweet potato seedlings treated with HaHS. Further investigation showed that H2S-mediated root formation was alleviated by N-1-naphthylphthalamic acid (NPA), an IAA transport inhibitor, and 2-(4-carboxyphenyl)4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), an NO scavenger. Similar phenomena in H2S donor-dependent root organogenesis were observed in both excised willow (Salix matsudana var. tortuosa Vilm) shoots and soybean (Glycine max L.) seedlings. These results indicated that the process of H2S-induced adventitious root formation was likely mediated by IAA and NO, and that H2S acts upstream of IAA and NO signal transduction pathways.

Key words: auxin; hydrogen sulfide; nitric oxide; root organogenesis; sweet potato.

Introduction

Root organogenesis is a significant event in higher plants. Adventitious rooting, a key step in clonal propagation, provides efficient anchoring on the substrate and facilitates uptake of water and nutrients from the soil. Adventitious root formation involves the development of meristematic tissue after removal of the primary root system. Adventitious root formation is a complex process that is affected by various environmental factors and multiple endogenous factors including phytohormones and other signaling molecules. The plant hormone auxins have been shown to promote rooting through the dedifferentiation of cells to reestablish the new apical meristem (Doerner 2008). Although a variety of components of auxin transport and signal transduction were identified, the molecular mechanism underlying the initiation of new root meristems is poorly understood (Berleth and Sachs 2001; Doerner 2008).
Nitric oxide (NO) is a diffusible multifunctional second messenger first described in animals, where it plays variable functions ranging from dilation of blood vessels to neurotransmission and defense during the immune response. Recent discoveries have implicated NO in root formation as well as in flowering, seed germination, senescence, maturity and programmed cell death. For example, it has been established that NO acts as a signal molecule in the hormonal cascade leading to root formation (Pagnussat et al. 2004) and is involved in Azospirillum brasilense-induced lateral root formation in tomato (Creus et al. 2005). Pagnussat et al. (2002, 2003) also demonstrated that NO mediates the auxin response which results in adventitious root formation (Pagnussat et al. 2002, 2003). Furthermore, a transient increase in NO concentration was observed in root formation on hypocotyl cuttings of cucumber (Cucumis sativus), showing that NO is required and is part of the molecular events leading to adventitious root development induced by indole acetic acid (IAA) (Pagnussat et al. 2002, 2003, 2004). Recently it was found that NO together with IAA increases the activities of calcium dependent protein kinases (CDPK) and phospholipase D during adventitious root formation in cucumber (Lanteri et al. 2006, 2008). Interestingly, many investigations demonstrated that NO acts downstream of carbon monoxide (CO), a known gaseous signaling molecule in animals, during adventitious root formation (Xu et al. 2006; Cao et al. 2007; Xuan et al. 2008).
Hydrogen sulfide (H2S) has also been found to be a “gaseous signal molecule” in animals in addition to NO and CO (Wang 2002). Hydrogen sulfide has been implicated in the induction of smooth muscle relaxation, hippocampal long-term potentiation, brain development, inflammation and antioxidant protection of neurons (Hosoki et al. 1997; Wang 2002; Kimura and Kimura 2004; Li et al. 2006). However, there has been little research in H2S biology in plants. In higher plants, H2S is evolved by enzymatic desulfhydration of cystein and metabolism of sulfite and sulfate (Rennenberg 1983, 1984). Hydrogen sulfide emission by higher plants has been observed in response to excess sulfur supply to roots or leaves (Sekiya et al. 1982; Rennenberg 1984, 1989). Short-term exposure of Brassica oleracea to atmospheric H2S resulted in a decrease in the activity of adenosine 5-phosphosulphate reductase in the shoot, and a threefold increase in thiol content occurred after 5 h exposure (Westerman et al. 2001). Hydrogen sulfide also serves as a signal molecule to control thiol levels in Arabidopsis thaliana (Riemenschneider et al. 2005). More recently, we demonstrated that H2S plays an important role in antioxidant metabolism counteracting abiotic stresses during wheat seed germination and sweet potato seedlings growth (Zhang et al. 2008, 2009). The previous researches mentioned above give a hint that the history of H2S in plants is just beginning.
In animals, it has already been demonstrated that the gaseous molecules NO, CO and H2S play important roles in cell signaling. Nitric oxide and CO have already been identified as signal molecules in plants and found to be involved in root formation (Pagnussat et al. 2002, 2003, 2004; Xu et al. 2006; Cao et al. 2007; Xuan et al. 2008). However, whether H2S acts as a second messenger similar to NO and CO in root organogenesis in plants remains unclear. In this report, we show evidence supporting a role for H2S in the regulation of root formation mediated by NO and IAA signaling in plants.

Results

Effect of H2S on adventitious root formation in sweet potato cuttings is dose dependent

Sodium hydrosulfide (NaHS), a H2S donor, which was applied to seedling cuttings of sweet potato (Ipomoea batatas) cultured in water solution, induced adventitious root organogenesis in a dose-dependent manner (Figure 1). This data shows that treatment with 0.2 mmol/L NaHS was the optimal concentration for induction of root number (Figure 1A) and root length (Figure 1B) compared to the control. However, at high concentrations of NaHS above 1.0 mmol/L, no additional stimulation of root number was found, rather partial inhibition was observed, indicating that higher level of NaHS could be toxic (Figure 1). Furthermore, application of different concentrations of H2S solutions obtained by diluting a saturated solution of H2S gave similar results on the number and length of roots. The optimal concentration of H2S was 0.5% saturated H2S for both root number and root length (Figure 1C,D).

Effect of NaHS on adventitious root formation is mediated through H2S

To verify the role of H2S in the promotion of adventitious root formation induced by NaHS, we used 0.2 mmol/L Na2S, Na2SO4, Na2SO3, NaHSO4, NaHSO3 and NaAC as controls for Na+ and for sulfur-containing components in our experimental system. These components were unable to induce adventitious root formation in seedling cuttings of sweet potato as effectively as 0.2 mmol/L NaHS (Figure 2). Application of the nonspecific H2S scavenger hemoglobin (Hb) alone did not affect adventitious root formation but Hb dramatically counteracted the effects of NaHS on root formation (Figure 2). From these data, we conclude that H2S or HS−, rather than other compounds directly or indirectly derived from NaHS, are responsible for the promotive effects of NaHS on adventitious root formation in sweet potato explants.

Changes in endogenous H2S, IAA and NO in sweet potato explants

To evaluate the roles of endogenous H2S, IAA and NO in root organogenesis, sweet potato explants were treated with NaHS or water and a time-course of changes in these compounds was carried out. A transient increase in endogenous H2S was observed in explants either treated with NaHS or water for 1 d, but the H2S content of NaHS-treated explants was significantly higher than that of controls. Thereafter, the levels of endogenous H2S in both explants fell significantly after 1 d, although higher levels of endogenous H2S was sustained in NaHS-treated explants till the end of experiment (8 d) when Treatment solutions were renewed every 12 h. After 8 d of treatment, adventitious roots (≥1 mm) were investigated and number of roots (A) and root length (B) values were expressed as mean ± SE (n = 30 explants from at least three independent experiments). Different letters above the bars indicate significant differences among treatments at the P < 0.05 level according to the least significant difference test.
compared to water control (Figure 3A). Changes in endogenous IAA and NO levels were similar to those for H2S but with different time-courses (Figure 3B,C). Transient increases in endogenous IAA and NO were observed followed by a decrease in IAA after 2 d and after 3 d for NO. The differing maxima in H2S, IAA and NO levels suggest that these molecules increase sequentially in response to NaHS application. Furthermore, explants treated with the NO donor sodium nitroprusside (SNP) maintained higher levels of endogenous H2S in comparison to control (Figure 3D), inferring that a feedback mechanism may be operating for the induction of root formation.

IAA and NO are involved in H2S-induced adventitious root organogenesis in sweet potato seedlings

Because our data show that endogenous IAA and NO levels increased in NaHS-treated explants, we investigated root organogenesis in sweet potato seedlings treated with the NO donor SNP or IAA in the presence of the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline1-oxyl-3-oxide potassium salt (cPTIO) and the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Sodium nitroprusside, IAA and NaHS treatment resulted in significant increment in adventitious root number (Figure 4A) and root length (Figure 4B). The NO scavenger cPTIO and IAA transport inhibitor NPA significantly inhibited adventitious root formation compared with the control (Figure 4). Application of cPTIO dramatically counteracted the promotive effects of the NO donor SNP as well as that of IAA and NaHS. On the other hand, the IAA transport inhibitor NPA could not result in alleviating the effects of both exogenous IAA and NO donor SNP. N-1-naphthylphthalamic acid was only effective in inhibiting the rooting in response to NaHS. Hemoglobin, which is a nonspecific scavenger for H2S and NO, played no positive role in endogenously-regulated root formation, but could significantly eliminate the promotive effects of exogenous H2S and NO donors. Hemoglobin could not alleviate the stimulatory effect of IAA on rooting, mainly because it is a large macromolecule that cannot easily enter into the plant (Figure 4).

Root organogenesis in willow and soybean stem cuttings is also induced by NaHS through NO and IAA signal pathways

Willow cuttings were also used to investigate adventitious root organogenesis induced by H2S. As shown in Figure 5(A,B), the number and the length of adventitious roots increased In this experiment, 0.2 mmol/L of sodium hydrosulfide (NaHS), 20 μmol/L 20 μmol/L of N-1-naphthylphthalamic acid (NPA), 100 μmol/L of 2-(4carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), 0.1 g/L hemoglobin (Hb), alone or combined were applied to the explants. Treatment solutions were renewed every 12 h. After 8 d of treatment, adventitious roots (≥1 mm) were investigated and number of roots (A) and root length (B) values were expressed as mean ± SE (n = 30 explants from at least three independent experiments). Different letters above the bars indicate significant differences among treatments at the P < 0.05 level according to the least significant difference test.
significantly after 8 d of treatment with appropriate NaHS concentration. The optimal concentration of NaHS was 0.2 mmol/L, and at higher concentrations its promotive effects disappeared. Figure 5(C) confirms the possibility that NO and auxin were involved in adventitious root organogenesis induced by H2S in willow. Treatment of willow explants with NaHS plus cPTIO or NPA resulted in a significant reduction in adventitious root formation induced by NaHS (Figure 5C). The induction of adventitious root formation by SNP and IAA in willow was also diminished by the NO scavenger cPTIO, but rooting was not affected by the auxin transport inhibitor NPA. Hemoglobin counteracted the promotive effects of exogenous NaHS and Cut shoot of willow was treated with 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mmol/L NaHS. Treatment solutions were renewed every 12 h. After 8 d of treatment, (A) a photograph of adventitious root formation in cut shoot of willow was taken and (B) root numbers (≥1 mm) was calculated and were expressed as mean ± SE (n = 30 explants from at least three independent experiments). (C) IAA and NO were involved in the H2S-induced formation of adventitious root in willow explants. In this experiment, 0.2 mmol/L of NaHS, 20 μmol/L of the IAA, 50 μmol/L of the NO donor sodium nitroprusside (SNP), 20 μmol/L of NPA, 100 μmol/L of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), 0.1 g/L hemoglobin (Hb), alone or combined, were applied to the explants. Root number were expressed as mean ± SE (n = 30 explants from at least three independent experiments). Different letters above the bars indicate significant differences among treatments at the P < 0.05 level according to the least significant difference test.
SNP on root formation. Furthermore, when applied alone, cPTIO and NPA significantly inhibited the formation of adventitious root compared to the control (Figure 5C), indicating that endogenous NO and auxin signals were involved in adventitious root formation.
Root induction and root growth in soybean cuttings in response to NaHS was qualitatively and quantitatively similar to what was observed in sweet potato and willow. Figure 6(A,B) show that exogenous NaHS promoted the formation and growth of adventitious roots and its effect was dose dependent. Compared with sweet potato and willow, lower concentrations of NaHS could promote lateral root organogenesis in soybean cuttings. Treatment of seedling explants with 0.01–0.04 mmol/L NaHS significantly enhanced the number of roots and the optimal concentration of NaHS was 0.02 mmol/L (Figure 6A,B). cPTIO eliminated the lateral root-promoting effects of the exogenous NO donor SNP as well as the promotive effects of IAA and NaHS, indicating that NO may function downstream in the auxin and H2S signal pathways for root formation (Figure 6C). The IAA transport inhibitor NPA could not block the lateral rootpromoting effect of SNP or IAA, but could counteract the effect of NaHS. Furthermore, when either NPA or cPTIO were applied to untreated soybean cuttings lateral root organogenesis was reduced relative to controls (Figure 6C), again suggesting that endogenous NO and IAA participated in lateral root formation.

Discussion

In this report, we show that the H2S donor NaHS, and aqueous solutions of H2S promoted formation and growth of lateral roots in a dose-dependent manner (Figure 1; Figure 5A, B; Figure 6A,B). Root initiation and growth could also be stimulated by treatment with the NO donor SNP and IAA (Figure 4; Figure 5C; Figure 6C). These effects were replicated both qualitatively and quantitatively in stem cuttings from three plant species, namely, sweet potato, soybean and willow. Sodium hydrosulfide has been widely used as a H2S donor in animals (Hosoki et al. 1997; Kubo et al. 2007). It dissolves in water and dissociates to produce Na+ and HS−; HS− associates with H+ and produces H2S. Other sulfur-containing components, such as S2−, SO42−, SO32−, HSO4−, HSO3− and Na+, which were used in this work as controls for NaHS but none were able to stimulate the formation of adventitious roots relative to controls (Figure 2). Furthermore, Hb, which can scavenge H2S, could counteract the promotive roles of NaHS on root formation (Figure 2; Figure 4; Figure 5C; Figure 6C). These results verify that H2S or HS−, rather than other compounds derived from NaHS, play a role in promoting adventitious root formation in plants. Hydrogen sulfide at higher concentration in plants is toxic and can block or slow down cell division activity in the apical meristem or change the distribution pattern of hormones. For example, excess H2S negatively affects plant growth by Soybean seedlings were treated with 0.0, 0.01, 0.02, 0.04, 0.06, 0.08 and 0.10 mmol/L NaHS. Treatment solutions were renewed every 12 h. After 8 d of treatment, (A) a photograph of root formation in seedlings of soybean was taken (treatments with 0.0 (CK) and 0.02 mmol/L NaHS (T) were compared in (A) and (B) root numbers (≥1 mm) was calculated and were expressed as mean ± SE. (C) IAA and NO were involved in the H2S-induced formation of adventitious root. In this experiment, 0.02 mmol/L of NaHS, 20 μmol/L of the IAA, 50 μmol/L of the NO donor sodium nitroprusside (SNP), 20 μmol/L of N-1-naphthylphthalamic acid (NPA), 100 μmol/L of 2-(4carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), 0.1 g/L hemoglobin (Hb), alone or combined, were applied to the explants. Root number were expressed as mean ± SE. Different letters above the bars indicate significant differences among treatments at the P < 0.05 level according to the least significant difference test.
inhibition of mitochondrial electron transport (Beauchamp et al. 1984; De Kok et al. 2002). In our experiments we have independent data showing that the effects of NaHS and H2S are unlikely to be toxic at the concentrations used. Measurement of plasma membrane integrity and malondialdehyde content (data not shown), which are two indicators of lipid peroxidation in cells show that NaHS concentrations of 1.0 mmol/L or less (for I. batatas and Salix matsudana) and 0.1 mmol/L or less (for Glycine max) used in our experiments caused no toxic effect on root or shoot cells. These evidences confirmed that NaHS promotes root formation in a way independent of “secondaryresponse”.
Adventitious root formation is a complex process involving an intricate network of signal molecules, one of them auxin. Auxin transport likely plays an important role in regulating hormone flux between IAA source and sink tissues, thereby influencing root development. Auxin induces dedifferentiation of parenchyma cells and entrance to cell division to form the root meristem (De Klerk et al. 1995). The action of auxin is coordinated with that of other plant hormones, such as gibberellin, abscisic acid, cytokinin, ethylene and other signaling molecules. For instance, Pagnussat et al. (2002) showed that the NO donors SNP and S-nitroso, N-acetyl penicillamine (SNAP), promoted adventitious root formation in cucumber seedlings, a similar response to that obtained with IAA. Subsequent investigations have demonstrated that NO is required for root organogenesis and acts downstream of the auxin response during in the rooting response (Pagnussat et al. 2003, 2004; Lanteri et al. 2006, 2008).
In this work, the NO scavenger cPTIO could completely prevent adventitious root formation induced by exogenous IAA (Figure 4; Figure 5C; Figure 6C), also confirming that NO is an intermediate in the auxin-regulated signaling cascade determining root morphology and physiology. Sodium hydrosulfide was able to mimic the stimulatory effects of IAA and of the NO donor SNP in inducing root organogenesis. The auxin polar transport inhibitor NPA reduced the stimulatory effect of H2S on adventitious root formation suggesting that auxins are also involved in the H2S-induced effect on rooting. Moreover, treatments with the NO scavenger cPTIO completely blocked H2S and IAA-induced adventitious root formation (Figure 4; Figure 5C; Figure 6C), indicating that NO is required for IAAor H2S-promoted adventitious root formation and is involved downstream of the H2S and IAA pathways. Interestingly, consistent with the results that IAA could induce transient increases in endogenous IAA and NO during the adventitious rooting process in cucumbers (Pagnussat et al. 2002), the exogenous H2S donor also induced a transient increase in endogenous H2S, IAA and NO in sweet potato explants and they functioned sequentially after application of the exogenous H2S donor NaHS (Figure 3A–C). These results also inferred that endogenous H2S was involved in the mechanism of adventitious root formation and it may act upstream of IAA and NO signal pathways.
It is interesting that NO serves as a downstream signal of H2S in root formation (Figure 4; Figure 5C; Figure 6C). Hydrogen sulfide can function as a reducing agent or an antioxidant, while NO is a redox agent in nature. They probably have adverse effects on protein cystein residues or on cellular thiol content. Furthermore, Kubo et al. (2007) demonstrated that NaHS/H2S could inhibit nitric oxide synthase and endothelial nitric oxide synthase activities in vitro most probably through interaction with BH4 and inhibit inducible nitric oxide synthase possibly through multiple unknown mechanisms, thereby downregulating NO levels in animal cells. On the other hand, published data obtained from animal tissues have shown that endogenous production of H2S from rat aortic tissues is enhanced by NO donor treatment (Zhao et al. 2001). The NO donor also enhances the expression level of cystathionine γ-lyase (CSE), which catalyzes L-cystein to H2S conversion in cultured vascular smooth muscle cells. Hosoki et al. (1997) observed that the vasorelaxant effect of NO donor SNP was enhanced by incubating rat aortic tissues with 30 μmol/L NaHS. On the contrary, in another study pre-treating aortic tissues with 60 μmol/L H2S inhibited the vasorelaxant effect of SNP. This paradox may be partially explained by the experimental conditions of these studies, including differences in tissue preparations and the development of tension before the application of H2S and NO. In addition, NO is mostly produced in endothelial cells and slightly in smooth muscle cells, while the production tissue source of H2S is smooth muscle cells but not endothelial cells (Wang 2002). It was deduced that in animal NO and H2S are effectively separated in cells and even compartmentalized in different sub-cellular locations.
The evidence in animals provided many hints for the similar mechanisms in plants, though cross-talk or networking between H2S, NO and other signals or regulators have yet to be elucidated. The data in this study support the hypothesis that NO is required for H2S-promoted adventitious root formation and that the three molecules H2S, IAA and NO are cellular messengers intimately involved with root formation. A serial linkage “H2S→IAA→NO→rooting” might be distinguished from the event. However, whether NO or IAA is involved in the feedback mechanism in root formation induced by H2S and how endogenous H2S is perceived and transduced into the specific responses needs further investigation. Apparently, the involvement of H2S in the signaling pathway of root organogenesis opens a more intricate field of research in the plant kingdom.

Materials and Methods

Plant material and chemicals

Sweet potato (Ipomoea batatas L., cv. Xushu 18) were supplied by Xuzhou Sweet Potato Research Center, Chinese Academy of Agricultural Sciences. Sweet potato seedlings were cut and selected for experiments. Seedling leaves were removed and the tips together with three fully expanded leaves from the top were left. Willow (Salix matsudana. var. tortuosa Vilm.) shoots were obtained from the horticulture center in Heifei University of Technology. Shoot with the tips and leaves removed were excised as for sweet potato and used for experiments. Seeds of soybean (Glycine max (L.) Merr.) obtained from Anhui Academy of Agricultural Sciences were surface-sterilized with 0.2% NaClO for 5 min, washed extensively and germinated in distilled water at 25 ± 1 ◦C for 3 d in the dark followed by 4 d in a 14 h photoperiod (3 000 lx). Soybean seedlings with their primary roots removed were used as explants for experiments. Sodium hydrosulfide and sodium nitroprusside (SNP, (Na2Fe(CN)5)·NO) purchased from Sigma (St Louis, MO, USA) were used as H2S and NO donors, respectively. N-1-naphthylphthalamic acid (NPA, Sigma), Hb (Sigma) and 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO, Sigma) were used as auxin polar transport inhibitor, non-specific H2S scavenger, and specific NO scavenger, respectively.

Treatments

To investigate the optimal effect of the H2S donor NaHS on root organogenesis in sweet potato seedlings and excised willow shoot, explants were cultured in Hoagland nutrient solutions (5 mmol/L Ca(NO3)2·4H2O, 1 mmol/L KH2PO4, 5 mmol/L KNO3, 2 mmol/L MgSO4·7H2O, 46.4 μmol/L H3BO3, 9.2 μmol/L MnCl2·4H2O, 1 μmol/L ZnSO4·7H2O, 0.32 μmol/L CuSO4·5H2O, 0.5 μmol/L Na2MoO4·2H2O, pH 6.0) containing 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mmol/L NaHS solutions, respectively. As for soybean seedlings, NaHS concentrations of 0, 0.01, 0.02, 0.04, 0.06, 0.08 and 0.1 mmol/L were used. To verify the hypothesis that NaHS-mediated root formation of the explants could be attributed to H2S or HS−, the non-specific H2S scavenger Hb, Na+, and sulfur-containing components, such as Na2S, Na2SO4, Na2SO3, NaHSO4, NaHSO3 and NaAC were used as the comparisons of the H2S donor NaHS. Explants of sweet potato seedlings used for experiments cultured in water (CK), NaHS at optimal concentration (obtained from above experiments), Na2S, Na2SO4, Na2SO3, NaHSO4, NaHSO3 or NaAC at the same concentration to NaHS, respectively. After 8 d of treatment, adventitious root number (≥1 mm) was calculated and were presented as mean ± SE (n = 30 explants from at least three independent experiments). In all experiments, the explants were maintained at 18 ± 1 ◦C in the dark and at 28 ± 1 ◦C in the light of 190 μmol/m2 per s (12 h/12 h), and all treatment solutions were renewed every 12 h. In this report, 20 μmol/L IAA, 50 μmol/L NO donor SNP, 20 μmol/L NPA, 100 μmol/L cPTIO, and 0.1 g/L Hb were applied to the explants.

Measurement of endogenous H2S in shoot tips

Hydrogen sulfide was determined by formation of methylene blue from dimethyl-p-phenylenediamine in H2SO4 according to the method described by Sekiya et al. (1982). Shoot tips (0.5 g) were ground and extracted in 5 mL phosphate buffer solution (pH 6.8, 50 mmol/L) containing 0.1 mol/L ethylene diamine tetra acetate and 0.2 mol/L ascorbic acid. The homogenate was mixed with 0.5 mL 1 mol/L HCl in a test tube to release H2S, and H2S was absorbed in zinc acetate (0.5 mL 1%) trap which is located in the bottom of the test tube. After 30 min of reaction, 0.3 mL 5 mmol/L dimethyl-p-phenylenediamine dissolved in 3.5 mmol/L H2SO4 was added into the trap. Then, 0.3 mL 50 mmol/L ferric ammonium sulfate in 100 mmol/L H2SO4 was injected in the trap. The amount of H2S in zinc acetate traps was determined spectrophometrically at 667 nm after leaving the mixture for 15 min at room temperature. Blanks were prepared by the same procedures with unused zinc acetate solution and known concentration of Na2S was used to make the calibration curve.

Determination of NO level

The NO level was measured according to the method of Murphy and Noack (1994). Shoot tips were incubated with 100 units of catalase and 100 units of superoxide dismutase for 5 min to remove endogenous reactive oxygen species before addition of oxyhemoglobin (10 mmol/L). After 3 min incubation, NO was quantified by spectrophotometric measurement of the conversion of oxyhemoglobin to methemoglobin.

Determination of IAA by high-performance liquid chromatography

Extraction and analysis of IAA in shoot tips of explants was carried out as described by Stefanˇ ciˇ c et al. (2007).ˇ

Statistical analysis

Significances were tested by one- or two-way ANOVA, and the results are expressed as the mean values ± SD of three independent experiments. Each experiment was repeated at least three times.

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