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Title: Hydrogen Sulfide Alleviates Aluminum Toxicity in Germinating Wheat Seedlings

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Running title: Hydrogen Sulfide Alleviates Aluminum Toxicity

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Article types: Research paper

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Authors: Hua Zhang

1, 2, *

, Zhu-Qin Tan 1, Lan-Ying Hu 1, Song-Hua Wang 3, Jian-Ping

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Luo 1, Russell L. Jones 2

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1

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Hefei, Anhui 230009, China;

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2

School of Biotechnology and Food Engineering, Hefei University of Technology,

Department of Plant and Microbial Biology, University of California, Berkeley,

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CA 94720, USA;

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3

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233100, China;

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*

Life Science College, Anhui Science and Technology University, Bengbu

Author for correspondence: Hua Zhang

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Tel: +86(0)-551 2901 506-8635, +1-510-280-4140;

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Fax: +86(0)551 2901 507;

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E-mail: .

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This work was supported by the Great Project of Natural Science Foundation from Anhui

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Provincial Education Department (ZD200910), the Natural Science Foundation of Anhui

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Province (070411009), and the innovation funding to undergraduate students at HFUT

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(XS08072, 0637).

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Hydrogen Sulfide Alleviates Aluminum Toxicity in Germinating Wheat

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Seedlings

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Abstract: Protective role of hydrogen sulfide (H2S) on seed germination and seedling

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growth was studied in wheat (Triticum) seeds subjected to aluminum (Al3+) stress. We

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show that germination and seedling growth of wheat is inhibited by high concentrations of

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AlCl3. At 30 mmol/L AlCl3 germination is reduced by about 50% and seedling growth is

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more dramatically inhibited by this treatment. Pre-incubation of wheat seeds in the H2S

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donor NaHS alleviates AlCl3-induced stress in a dose-dependant manner at an optimal

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concentration of 0.3 mmol/L. We verified that the role of NaHS in alleviating Al3+ stress

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could be attributed to H2S/HS- by showing that the level of endogenous H2S increased

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following NaHS treatment. Furthermore, other sodium salts containing sulfur were

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ineffective in alleviating Al3+ stress. NaHS pretreatment significantly increased the

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activities of amylases and esterases and sustained much lower levels of MDA and H2O2 in

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germinating seeds under Al3+ stress. Moreover, NaHS pretreatment increased the activities

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of guaiacol peroxidase, ascorbate peroxidase, superoxide dismutase and catalase and

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decreased that of lipoxygenase. NaHS pretreatment also decreased the uptake of Al3+ in

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AlCl3-treated seed. Taken together these results suggest that H2S could increase antioxidant

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capability in wheat seeds leading to the alleviation of Al3+ stress.

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Key words: aluminum stress; antioxidant enzymes; hydrogen sulfide; seed germination

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and seedling growth; wheat (Triticum).

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Hydrogen sulfide (H2S) has recently been identified as a third endogenous gaseous

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transmitter after nitric oxide (NO) and carbon monoxide (CO) in animals where it plays

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various roles ranging from regulation of the nervous and cardiovascular systems (Wang

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2002; Li et al. 2006; Yang et al. 2008). For example, Hosoki et al. (1997) demonstrated that

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H2S acts synergistically with NO to regulate smooth muscle relaxation. It is now well

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established that NO is involved in diverse physiological processes in plants (Delledonne

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2005). More recent evidence has been accumulated that CO is also involved in different

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biological process in plants such as root formation (Cao et al. 2007) and protection against

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oxidative damage induced by salinity (Huang et al. 2006; Xu et al. 2006) and mercury (Han

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et al. 2007). Plants are known to synthesize and release H2S. For example, H2S was proved

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to be released from leaves of cucumber (Cucumis sativus L.), sqush and pumpkin

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(Cucurbita pepo L.), cantaloupe (Cucumis melo L.), corn (Zea mays L.), soybean (Glycine

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max [L.] Merr.) and cotton (Gossypium hirsutum L.) (Wilson et al. 1978). H2S is also

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produced by cut branches, detached leaves, leaf discs, or tissue cultures, evidence that green

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cells of higher plants can release H2S into the atmosphere (Wilson et al. 1978; Winner et al.

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1981; Sekiya et al. 1982a, 1982b; Rennenberg 1983, 1984; Rennenberg et al. 1990). It is

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conceivable that H2S might serve as an informational signal to other parts of the plant, or to

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plants in the vicinity in a similar manner to NO and CO (Zhang et al. 2008a; Zhang et al.

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2009a, b).

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Recently, several types of specific desulfhydrases have been identified and

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functionally characterized in plants, confirming that H2S might be released by the action of

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desulfhydrases localized in different cellular compartments such as the cytosol,

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mitochondria and plastids (Leon et al. 2002; Riemenschneider et al. 2005b; Rausch and

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Wachter 2005). Although the individual roles of these enzymes are not yet understood,

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L-cysteine desulfhydrase expression and activity are induced upon pathogen attack,

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suggesting that released H2S has a role in plant defense (Bloem et al. 2004; Rausch and

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Wachter 2005). Riemenschneider et al. (2005a) also investigated that impact of elevated

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H2S on metabolite levels, the activity of enzymes and expression of genes involved in

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cysteine metabolism, confirming that H2S serves as a signal molecular to control thiol

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levels in Arabidopsis thaliana. Although H2S emission has been widely observed in many

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plant species its role as a signaling molecule in plants has yet not to be clearly defined.

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Nevertheless, H2S is known to play a role in various responses of plants against stresses.

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Roles of H2S in response to pathogen attack (Bloem et al. 2004), Cu2+ tolerance in

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germinating wheat seeds (Zhang et al. 2008a), osmotic stress tolerance in sweet potato

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seedlings (Zhang et al. 2009b), SO2 tolerance in pine trees (Hällgren and Fredriksson 1982;

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Sekiya et al. 1982b), and freezing tolerance in wheat shoots (Stuiver et al. 1992), all

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suggest that H2S is involved in mechanisms of plant resistance.

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Aluminum ions (Al3+) are ubiquitous in soil and are especially toxic in acidic soils.

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Furthermore, Al3+ is one of the major limiting factors affecting crop production in tropical

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regions having acid soils (Foy 1984; MacDonald and Martin 1988). It is estimated that 70%

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of tropical soil in the Americas are acidic and have toxic levels of Al3+ that are growth

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limiting for crops (Marschner 1995). One of the primary causes of Al3+ toxicity is oxidative

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stress due to accumulation of reactive oxygen species (ROS), such as the superoxide anion

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(O2·¯) and hydrogen peroxide (H2O2). These ROS have been shown to bring about lipid

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peroxidation in soybean root tips (Horst et al. 1992), detached rice leaves (Kuo and Kao

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2003), and in roots of Cassia tora L. (Wang and Yang 2005) and Melaleuca trees (Tahara et

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al. 2008). Our previous report on Al3+-induced oxidative stress in wheat seedlings

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confirmed that ROS are a key component of the response of wheat to Al3+ (Zhang et al.

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2008b). Production and removal of ROS generally involve non-enzymatic and enzymatic

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antioxidant systems and if the equilibrium between ROS production and breakdown fails,

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oxidative damage occurs (Apel and Hirt 2004). It is now becoming increasingly clear that

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plant resistance to Al3+-induced oxidative stress involves a wide range of signaling

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molecules, such as Ca2+/CaM, inositol 1,4,5-triphosphate (IP3), salicylic acid, H2O2 and NO

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(Jones and Kochian 1995; Wang et al. 2004; Wang and Yang 2005; Zheng and Yang 2005;

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Zhang et al. 2008b).

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In this paper we describe the effects of H2S pretreatment on the response of wheat

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seeds to Al3+ toxicity. In previous work, we demonstrated that H2S promotes seed

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germination and root formation, and acts as an antioxidant signal counteracting Cu2+ and

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osmotic stress in plants (Zhang et al. 2008a; Zhang et al. 2009a, b). We now show that H2S

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pretreatment offers significant protection against the toxic effects of Al3+. Our data show

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that there is a strong correlation between reduced ROS level and lowered lipid peroxidation

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and pretreatment of wheat seeds with H2S. We concluded that H2S acts as an antioxidative

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signaling molecule that participates in alleviation of Al3+ toxicity during wheat seed

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germination.

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Results

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Inhibition of germination and seedling growth in wheat by Al3+

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The inhibitory effects of Al3+ on wheat seed germination and wheat seedling growth and

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development were examined over a wide range of AlCl3 concentrations from 5 mmol/L to

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150 mmol/L. Wheat seeds are relatively insensitive to concentrations of AlCl3 of 5 mmol/L

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or below (Table 1 and data not shown). Wheat seed germination percentage was slightly

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higher in 5 mmol/L AlCl3 relative to controls germinated in H2O, and at 10 mmol/L AlCl3

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germination was identical to that in H2O (Table 1). Coleoptile length and radicle number

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were also unaffected by 5 mmol/L AlCl3, but radicle length was inhibited by 20%.

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To test the alleviating effects of H2S on wheat germination and growth we determined

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the concentration of AlCl3 which inhibited germination by 50%. As Table 1 and Figure 1

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show a concentration of 30 mmol/L AlCl3 inhibited germination by 51% and radicle and

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coleoptile length were reduced by 95% and 40% respectively. Radicle number in seeds

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incubated in 30 mmol/L was reduced to 25% of controls in H2O (Table 1). Concentrations

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of AlCl3 above 60 mmol/L inhibited radicle growth almost completely, but even at this

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concentration of AlCl3 coleoptile growth was still observed. Based on these data we chose a

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working concentration of 30 mmol/L AlCl3 to study the ameliorating effects of H2S.

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Amelioration of Al3+ stress in wheat by the H2S donor NaHS

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We first established whether the H2S donor NaHS had toxic effects on wheat seed

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germination and seedling growth. Wheat seeds were germinated for 36 h in increasing 5

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NaHS concentrations from 0.3 mmol/L to 1.5 mmol/L (Table 2 and Figure 2A). There was

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no statistically significant effect of NaHS relative to controls in H2O on any of the

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parameters that we measured. Germination, coleoptile and radicle elongation, and radicle

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number was essentially unchanged following incubation in NaHS relative to control seeds

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incubated in water for 36 h.

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We next tested the ability of NaHS to alleviate the toxic effects of AlCl3 by pre-treating

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wheat seeds with the H2S donor for 12 h prior to incubation with Al3+. To establish the

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effective concentration of NaHS in alleviating the effects of AlCl3, the H2S donor was

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applied to wheat seeds stressed with 30 mmol/L AlCl3 in the range of 0.3 to 1.5 mmol/L

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(Table 3 and Figure 2B). NaHS pretreatment was effective in alleviating the toxic effects of

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Al3+ at all concentrations that were tested, but the optimal NaHS concentration for

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alleviating germination and seedling growth was 0.6 mmol/L. At this concentration

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germination percentage was increased by 74%, radicle number by 80%, and radicle and

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coleoptile length by 202% and 255% respectively. Clearly, the H2S donor exerted a strong

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positive effect on wheat germination and seedling growth following exposure to Al3+.

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To verify the role of H2S in the promotion of seed germination induced by NaHS in

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Al3+ treated wheat we tested the effect of a range of sodium salts including those that

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contained sulfur. Seeds were pre-incubated in 0.6 mmol/L Na2S, Na2SO4, Na2SO3, NaHSO4,

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NaHSO3, and NaAC for 12 h and incubated for a further 48 h in 30 mmol/L AlCl3. As the

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data in Figure 2C show, only NaHS was able to overcome the toxic effects of Al3+. There

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was no significant effect of the other sodium salts on germination of wheat seeds. From

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these results it is concluded that either H2S or HS-, rather than other compounds derived

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from NaHS, are responsible for the alleviating Al3+ stress in wheat seedlings.

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At millimolar concentrations, NaHS solutions are neutral to basic, the pH of NaHS

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solutions increase from pH 6.85 at 0.01 mmol/L to pH 8.69 at 4 mmol/L. We chose a

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working concentration of 0.6 mmol/L NaHS for our experiments. In order to prove that the

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promotive role of NaHS pretreatment on seeds germination against Al3+ stress is

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contributed to NaHS rather than the pH value due to NaHS added in the pretreatment

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solutions, 0.6 mmol/L PBS (Na2HPO4-NaH2PO4 buffer) at different pH value (5.8, 6.0, 6.2,

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6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.8, 8.0) were used as the controls of NaHS with different

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concentration (0, 0.3, 0.6, 0.9, 1.2, 1.5 mmol/L). Wheat seeds were pretreated with water

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control (CK), 0.6 mmol/L NaHS, and 0.6 mmol/L Na2HPO4-NaH2PO4 buffer solutions at

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different pH value 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.8, and 8.0, respectively. After

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12 h of pretreatment, seeds were washed with water and then subsequently subjected to

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30.0 mmol/L AlCl3 stress for a further 48 h. Figure 2D showed that the strong positive

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effect of H2S donor on seed germination and seedling growth against Al3+ stress is

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independent on pH value, because pretreatments with PBS buffer (pH 5.8~8.0) did not

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promote seed germination under Al3+ stress as NaHS pretreatments did (Figure 2D).

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Effect of NaHS pretreatment on the activities of amylase and esterase

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Whereas hydrolytic enzymes such as α-amylases and esterases are unlikely to be required

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for germination of what seeds, seedling growth and development are known to depend on

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the mobilization of stored endosperm reserves. We therefore examined the activities of

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amylases and esterase in wheat seeds exposed to AlCl3 following pretreatment with and

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without NaHS (Figure 3 and 4). Hydrolases were extracted from dry wheat seeds (CK0’),

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from seeds pretreated in water or NaHS for 12 h (12’/0) and from seeds incubated for up to

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48 h in 30 mmol/L AlCl3 (12~48). Amylase activity was determined following

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electrophoresis (Figure 3A) or by measurement of enzyme activity colorimetrically (Figure

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3B). Amylase activity was not detected following electrohporesis of extracts from dry

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seeds and activity was low when it was measured colorimetrically. Pretreatment of seeds

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with H2O or NaHS for 12 h caused an increase in amylase activity but the increase was

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much greater in the presence of NaHS. The H2S donor was effective in ameliorating the

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effect of AlCl3 on the activity of amylase and its activity increased linearly for the first 36 h

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of incubation. There was a much smaller increase in amylase activity in response to AlCl3

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in seeds pretreated in H2O.

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There was a much less pronounced effect of NaHS on esterase activity (Figure 4A

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and B). Esterase activity was high in dry seeds and was relatively unchanged by incubation

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in H2O for 12 h or following treatment with AlCl3. NaHS pretreatment for 12 h did not

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increase esterase activity, but there was a small and statistically significant effect of NaHS

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on esterase activity when seeds were incubated for up to 48 h in 30 mmol/L AlCl3. This

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stimulation of esterase activity by the H2S donor was observed following eletrophoresis

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(Figure 4A) and by colorimetric measurement of esterase activity (Figure 4B).

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Effect of NaHS pretreatment on contents of H2O2, O2·¯ and MDA

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Malondialdehyde (MDA) has been widely used to estimate the extent of lipid peroxidation

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in plant tissues and we used this assay to determine the effect of NaHS treatment in wheat

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seeds exposed to AlCl3 (Figure 5A). MDA-reactive lipids were low in dry seeds and

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increased almost two-fold following incubation for 12 h in either H2O or NaHS. There is a

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dramatic increase in MDA-reactive lipids following 12 h incubation in AlCl3 in seeds

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pretreated with H2O, but the increase was dramatically lower in seeds pre-incubated in

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NaHS (Figure 5A). The amount of MDA-reactive lipids increased in seeds exposed to

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AlCl3 for 24 h, 36 h and 48 h, but the H2S donor dramatically reduced their amount relative

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to seeds pre-incubated in H2O.

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Next, we measured the accumulation of H2O2 and O2- in wheat seeds pretreated with

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the H2S donor then exposed to Al3+ stress (Figure 5B, C). The concentrations of H2O2 and

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O2- were both low in dry wheat seeds and they increased about two-fold following

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incubation in either H2O or NaHS for 12 h. Exposure of H2O-pretreated seeds to AlCl3

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brought about more than a three-fold increase in ROS species, but following pretreatment

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with NaHS for 12 h the increase in H2O2 and O2- was greatly reduced. Thus H2O2 levels

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increased about 50% and O2- increased 60% in AlCl3 following NaHS pretreatment,

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whereas

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(Figure 5B, C).

H2O pretreatment increased H2O2 and O2- content by more than 200% after 12 h

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Effects of NaHS pretreatment on SOD, CAT, APX, POD and LOX activities

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We next investigated whether reduced levels of ROS in seeds pretreated with NaHS might

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result from an increase in the activity of ROS-scavenging enzymes in AlCl3 pretreated

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seeds (Figure 6). The activities of all ROS scavenging enzymes were low in dry wheat

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seeds and they increased following incubation for 12 h in either H2O or NaHS. The

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activities of SOD and APX fell significantly during the first 12 h of incubation of H2O

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pretreated seeds in AlCl3. By contrast SOD activity did not change following Al3+

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treatment of NAHS-pretreated seeds (Figure 6A), but the activity of APX increased by

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about 50% in these seeds (Figure 6C).

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Catalase and peroxidase activities were not markedly affected by AlCl3 treatment in

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seeds pretreated with H2O, but these two enzymes showed an increase in their activities

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during 36 h of incubation in NaHS. Thus CAT activity increased almost two-fold whereas

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POD actvity increased about three-fold in AlCl3 treated seeds pretreated with NaHS

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(Figure 6B, D).

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We also assayed the activity of lipoxygenase, the enzyme that gives rise to molecules

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such as jasmonic acid. LOX activity was low in dry seeds and increased only slightly on

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further incubation in H2O or NaHS for 12 h (Figure. 6E). LOX activity continued to

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increase in H2O pretreated seeds incubated in AlCl3 and by 24 h after exposure to Al3+

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LOX activity was more than 300% higher than in seeds pretreated with H2O for 12 h but

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there was a dramatic decline in LOX activity in these seeds when incubated in AlCl3 for 36

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h and 48 h. By 48 h after AlCl3 treatment the activity of LOX returned to the level found in

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seeds pretreated in H2O for 12 h (Figure 6E). LOX activity in NaHS pretreated seeds also

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increased following Al3+ exposure but the increase was much less than that observed in

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seeds pretreated with H2O and there was a similar decline in LOX activity after 36 h of

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incubation.

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NaHS pretreatment reduces Al accumulation in wheat seedlings

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We measured the accumulation of Al in wheat seeds and seedlings under the conditions of

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our germination experiments. As expected the Al content in dry seeds and 12 h imbibed

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seeds was low, but following incubation in AlCl3 Al concentrations increased (Figure 7).

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The increase in Al was much more dramatic in seeds pre-incubated for 12h in H2O

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compared to those pre-incubated in NaHS. After 48 h incubation in AlCl3, the Al

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concentration in H2O pretreated seeds was more than twice that found in seeds

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pre-incubated in NaHS (Figure 7).

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Endogenous H2S content is elevated in wheat seedlings treated with NaHS

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We measured H2S concentration in wheat seeds under the various conditions of incubation

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(Figure 8). Endogenous H2S was low in dry seeds, but it increased following incubation for

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12 h. As expected, the increase in H2S was much greater in NaHS-treated seeds. AlCl3

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treated seeds showed a sharp increase in H2S during the first 12 h and thereafter there was

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no change in its concentration. There was also an increase in H2S levels in H2O-pretreated

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seeds after 12 h incubation in AlCl3, but thereafter the amount of H2S declined reaching the

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level found in seeds pre-incubated in H2O for 12 h. These results show convincingly that

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the H2S donor NAHS donor contributes to an increase in endogenous H2S concentrations in

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wheat seedling tissues.

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Discussion

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Aluminum is abundant in soils and in acidic soils it is often present at concentrations that

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are inhibitory to plant growth and development. In this paper we show that the inhibitory

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effects of AlCl3 on germination and seedling growth in wheat can be alleviated by

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pretreatment with the H2S donor NaHS. Our data show that AlCl3 at 30 mmol/L inhibits

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wheat seed germination by about 50% and coleoptile and radicle growth is even more

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severely inhibited at this concentration of Al3+. Preincubation of wheat seeds in NaHS for

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12 h alleviates the toxic effects of AlCl3. Pretreatment of wheat seeds with NaHS also

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increases the production of ROS scavenging enzymes and reduces the oxidation of lipids.

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NaHS is a commonly used H2S donor in biological systems (Hosoki et al. 1997).

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NaHS dissociates to Na+ and HS- in solution and HS- associates with H+ to produce H2S.

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That NaHS functions as an H2S donor, alleviating the effects of Al3+ in wheat is supported

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by two lines of evidence. First, among the solutions that we tested, only NaHS effectively

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reverses the toxic effects of Al3+ in wheat and its ameliorating role was independent on the

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pretreatment of pH value changes. Solutions of Na2S, Na2SO4, Na2SO3 NaHSO4, and

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NaHSO3 were largely ineffective in stimulating germination or seedling growth in 10

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AlCl3-treated wheat. Second, wheat seeds exposed to NaHS show a dramatic increase in

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extractable H2S levels after incubation in this H2S donor (Figure 8). Seeds incubated in

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H2O also show a significant increase in endogenous H2S but this is likely to be derived

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from the mobilization of sulfur containing storage proteins in the wheat grain (Shewry

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1995).

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In accord with previous observations (Zheng and Yang 2005) our data show that Al3+

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stress in plants results in overproduction of ROS. We show that AlCl3 brings about a

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dramatic increase in H2O2 and O2- levels and that these increases are mitigated by

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pretreatment with NaHS. Furthermore, NaHS pretreatment increases the activities of

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enzymes that can scavenge ROS including APX, CAT, POD and SOD. The net result of the

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increase in ROS scavenging enzymes is a dramatic reduction on lipid oxidation as shown

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by much lower amounts of MDA-reactive lipids in NaHS pretreated wheat. Taken together

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this data strongly supports the idea that NaHS alleviates Al3+ stress by reducing ROS levels.

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It is well established in plants that the reactive nature of ROS makes them potentially

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harmful to all cellular components. Plants have evolved the capacity to eliminate ROS with

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an efficient scavenging system and enzymes such as APX, CAT and SOD are among the

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most effective ROS scavengers (Van Breusegem et al. 2001; Mittler 2002, 2006). In our

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study, overproduction of O2·¯, H2O2 in Al3+ stressed wheat (Figure 5) might contribute to

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the lipid peroxidation and cellular membrane damage, which in turn results in inhibition of

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seed germination and seedling growth (Table 1 and Figure 1). H2O2 is produced in response

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to various stimuli in plants (Bowler and Fluhr 2000) and acts as a signal for the activation

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of stress-response and defense pathways (Bartosz 1997; Blokhina et al. 2003). Thus, H2O2

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can be viewed as cellular indicator of stress and as a second messenger involved in signal

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transduction pathways linked to stress (Knight and Knight 2001). Whether H2S works in

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concert with H2O2 as a second messenger in stress responses such as that brought about by

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Al3+ remains to be elucidated.

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In summary, our data strongly support the hypothesis that the H2S donor NaHS

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alleviates Al3+ toxicity in wheat by suppressing the production of ROS. The H2S donor

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increases the activities of ROS scavenging enzymes that bring about a reduction in H2O2

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and O2- leading to a reduction in lipid peroxidation and membrane damage. These data

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have implications for studies of metal and other stresses in plants and our goals are to

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understand the details underlying the H2S signal transduction pathway.

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Our data also provide evidence that pretreatment with exogenous H2S donor leads to

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higher levels of endogenous H2S signal and that this higher H2S concentration alleviates

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Al3+-induced oxidative damage to germinating seed. This phenomenon was correlated with

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the suppression of H2O2 and MDA overproduction in NaHS-pretreated seeds under Al3+

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stress. The H2S donor-induced lowering of H2O2 and MDA levels could be attributed to the

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increased activities of ROS-scavenging enzymes and the decreased activity of LOX. Many

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phenomena such as expression of L-cysteine desulfhydrase upon pathogen attack (Bloem et

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al. 2004), freezing tolerance affected by H2S fumigation (Stuiver et al. 1992), and emission

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of H2S from plant against SO2 injury (Hällgren and Fredriksson 1982; Sekiya et al. 1982b),

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indirectly support our conclusions that H2S might activate an H2O2-mediated antioxidant

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signaling pathway and play a protective role in plant defense. Our evidence adds to the

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concept that H2S can be another important signal molecule for abiotic stress tolerance.

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Genetic analysis and further physiological studies will help establish H2S as a player in

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signal in transduction cascades in plants and improve our understanding of the mechanisms

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of H2S perception and how it is transduced into specific downstream responses.

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Materials and methods

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Materials and treatments

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Wheat (Triticum aestivum L., Yangmai 158) seeds were supplied by the Jiangsu Academy

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of Agricultural Sciences, Jiangsu Province, China. Sodium hydrosulfide (NaHS, Sigma)

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was used as hydrogen sulfide (H2S) donor according to Hosoki et al. (1997). Wheat seeds

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were sterilized with 0.1% HgCl2 for 3 min, washed extensively with dH2O, and dried with

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filter papers. To establish the inhibitory effect of AlCl3 on germination and seedling growth,

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sterilized wheat seeds of approximately equal size were selected and allocated randomly in

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Petri dishes (9 cm diameter × 1.2 cm depth, 50 seeds per dish) and germinated in the dark at

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℃ with 0, 5, 10, 15, 20, 25, 30, 60, 90, 120, 150 mmol/L AlCl , for 48 h. Seeds were

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recorded as germinated when the length of the coleoptile or radicle reached 50% of the

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length of seed. The length of coleoptiles and radicles and radicle number were also

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recorded. The protective roles of H2S on the Al3+-induced inhibition of seed germination

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and seedling growth was examined by pre-treating seeds with 0, 0.3, 0.6, 0.9, 1.2, 1.5

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mmol/L NaHS respectively for 12 h, and subsequently subjecting them to a semi-inhibitory

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AlCl3 concentration. To verify that NaHS alleviated Al3+ stress via the production of H2S or

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HS-, various sodium salts were tested including Na2S, Na2SO4, Na2SO3 NaHSO4, NaHSO3,

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and NaAC. Seeds were pretreated for 12 h with water (CK), optimal concentration of NaHS

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obtained from the above experiment or with Na2S, Na2SO4, Na2SO3 NaHSO4, NaHSO3, or

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NaAC at the same concentration as NaHS, and then subjected to the semi-inhibitory Al3+

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stress for a further 48 h and germination percentage and growth measured. To test whether

343

the possible positive effect of H2S donor on seed germination against Al3+ is dependent on

344

the pretreatment of pH value or not, which is resulted from NaHS pretreatment with

345

different concentration (0, 0.3, 0.6, 0.9, 1.2, 1.5 mmol/L) added in the solutions,

346

Na2HPO4-NaH2PO4 buffer solutions at different pH value 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2,

347

7.4, 7.8, 8.0 were use as the controls of different concentration of NaHS (0, 0.3, 0.6, 0.9,

348

1.2, 1.5 mmol/L). Wheat seeds were pretreated with water (CK), 0.6 mmol/L NaHS, and

349

0.6 mmol/L Na2HPO4-NaH2PO4 buffer solutions at different pH value 5.8, 6.0, 6.2, 6.4, 6.6,

350

6.8, 7.0, 7.2, 7.4, 7.8, and 8.0, respectively. After 12 h of pretreatment with 0.6 mmol/L

351

NaHS and and 0.6 mmol/L Na2HPO4-NaH2PO4 buffer solutions at different pH value, seeds

352

were washed with water and then subsequently subjected to 30.0 mmol/L buffered AlCl3

353

stress for a further 48 h; and germination percentage and growth are measured. The AlCl3

354

treatment solution was replaced by a fresh batch every 12 h and geminating seeds (0.5±0.05

355

g) were randomly selected every 12 h and sampled for analysis.

3

356 357

Enzyme assays and electrophoretic analysis

358

The activity and electrophoretic analysis of amylase were preformed as described by Zhang

359

et al. (2005). Wheat seeds (0.5±0.05 g) were homogenized in 5 mL ice-cold phosphate

13

360

buffer (200 mmol/L, pH 8.3, containing 1% PVP), the homogenate was centrifuged at

361

12,000 g for 30 min and the supernatant was used as amylase preparations for further assay.

362

Total amylase activity was determined using the starch-iodine method and one unit of

363

activity (U) was calculated as the amount of the enzyme required to reach 50% of initial

364

color intensity. Electrophoresis was performed in 10% vertical polyacrylamide gels. Fifteen

365

μl of amylase preparation was applied per well. To visualize the bands of amylase, the gel

366

was incubated in 50 mmol/L PBS (pH 7.0) containing 1% boiled soluble starch at 25

367

30 min. After being washed with distilled water for 3 times, the gel was stained with 0.6%

368

I2 and 6% KI solution.

℃ for

369

Native PAGE and activity determination of esterases (EC 3.1.1.3) were assayed

370

following the methods of Deising et al. (1992). To visualize the bands of esterase activities,

371

gel was washed twice for 20 min in 100 mmol/L Tris-HCl, pH 8.0. The indoxyl acetate

372

substrate (35 mg) was dissolved in 1 mL of acetone and added to 49 mL of 100 mmol/L

373

Tris-HCl, pH 8.0. Gels were incubated with the solution by constant agitation at room

374

temperature until bands of desired intensity appeared. Esterase activity was assayed at 30

375

by measuring the hydrolysis of p-nitrophenyl butyrate at 400 nm. Reaction mixtures

376

consisted of 600 μl of Tris-HCl buffer (100 mmol/L, pH 8.0), 200 μl of an enzyme

377

preparation, and 200 μl of a stock solution of 37.5 mmol/L p-nitrophenyl butyrate in the

378

same buffer.



379 380

Determination of MDA, H2O2 and O2·¯

381

The contents of MDA-reactive lipids, H2O2 and O2·¯ were determined by the procedures

382

described by Wang et al. (2004). Wheat seeds were ground in 3 mL of 0.1% trichloroacetic

383

acid (TCA) solution. The homogenate was centrifuged at 15 000 g for 10 min and 0.5 mL

384

of the supernatant fraction was mixed with 2 mL 20% TCA containing 0.5% thiobarbituric

385

acid (TBA). The mixture was heated at 90

386

10,000 g for 5 min. The absorbance was recorded at 532 nm and the value for non-specific

387

absorption at 600 nm was subtracted. The extinction coefficient of 155 mmol L-1 cm-1 was

388

used to calculate the content of MDA.

℃ for 20 min, cooled and then centrifuged at

14

389

For determination of H2O2, wheat seeds were ground and extracted in 3 mL cold

℃ for 20 min. Then, 0.5 mL of

390

acetone. The homogenate was centrifuged at 10,000 g at 4

391

the supernatant fraction was mixed with 1.5 mL of CHCl3 and CCl4 (1:3, V/V) mixture.

392

Subsequently 2.5 mL of distilled water was added and the mixture centrifuged at 1,000 g

393

for 1 min and the aqueous phase collected for H2O2 determination. The reaction system

394

included 0.5 mL sample, 0.5 mL of buffer (PBS, 200 mmol/L, pH 7.8), and 20 μL 0.5 unit

395

of catalase (as controls) or inactive catalase protein (catalase was inactivated by incubating

396

in boiled water for 5 min,). After the mixture was incubated at 37

397

200 mmol/L Ti-4-(2-pyridylazo) resorcinol (Ti-PAR) was added. Then the reaction

398

mixtures were incubated at 45

399

measured.

℃ for 10 min, 0.5 mL of

℃ for another 20 min. The absorbance at 508 nm was

400

O2·¯ content was calculated by an extinction coefficient of 2.16×104 M–1 cm–1. Seeds

401

(0.5±0.05 g) were ground with 3 mL of 50 mmol/L Tris–HCl buffer (pH 7.5). The

402

homogenate was centrifuged at 5,000 g at 4°C for 10 min. The reaction mixture (1 mL)

403

contained

404

3’-(1-(phenylamino-carbonyl)-3,4-tetrazolium-

405

acid hydrate) and 50 μL of sample extracts. Corrections were made for the background

406

absorbance in the presence of 50 U of SOD.

50

mmol/L

Tris–HCl

buffer

(pH

7.5),

0.5

mM

bis(4-methoxy-6-nitro)

XTT

(sodium,

benzenesulfonic

407 408

Assays of LOX, SOD, CAT, APX, POD activities

409

Activity of lipoxygenase (LOX, EC 1.13.11.12) was determined following the description

410

by Surrey (1964) and those of superoxide dismutase (SOD, EC 1.1.5.1.1), catalase (CAT,

411

EC1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), guaiacol-dependent peroxidase

412

(POD, EC 1.11.1.7) were assayed according to García-Limones et al. (2002). Wheat seeds

413

were homogenized in ice-cold 50 mmol/L phosphate buffer (pH 7.8) containing 1.0

414

mmol/L EDTA. The homogenate was centrifuged at 15,000 g at 4

415

supernatant was used for activity measurement. The activities of APX were determined in

416

the presence of 0.5 mmol/L ascorbic acid and 0.5 mmol/L H2O2 by monitoring the decrease

417

in absorbance at 290 nm. Activities of CAT were determined spectrophotometrically by

℃ for 10 min. The

15

418

monitoring the decrease in absorbance at 240 nm. Activities of SOD were assayed by

419

measuring its capacity of inhibiting the photochemical reduction of nitro-blue tetrazolium.

420

Analysis of guaiacol POD capacity was based on the oxidation of guaiacol using hydrogen

421

peroxide. The reaction mixture contained 2.5 mL of 50 mmol/L potassium phosphate buffer

422

(pH 6.1), 1 mL of 1% hydrogen peroxide, 1 mL of 1% guaiacol and 10–20 μL of enzyme

423

extract. The increase in absorbance at 420 nm was read.

424 425

Al content determination

426

Seed samples (0.5 g) were ground in mortars with 5 mL deionized water, then the

427

homogenate was digested at 80

428

(1:1:3). After evaporation, the residue was mixed with 2% (v/v) nitric acid and the volume

429

was increased to 5 mL with deionized H2O and Al content was quantified by atomic

430

absorption spectrophotometer using a Hitachi Model 180/80 flame (180–80 Hitachi, Tokyo,

431

Japan) and graphite furnace (Hitachi 180/078) atomic absorption spectrophotometer, and an

432

automatic data processor were used for measurement.

℃ with a mixture of HNO , H O , and deionized H O 3

2

2

2

433 434

Measurement of endogenous H2S in seeds

435

H2S was determined by formation of methylene blue from dimethyl-p-phenylenediamine in

436

H2SO4 as described previously (Zhang et al. 2008a).

437 438

Statistical analysis

439

Significances were tested by one-way or two-way ANOVA, and the results are expressed as

440

the mean values ± SD of three independent experiments. Each experiment was repeated at

441

least three times.

442 443

Acknowledgments

444

This work was supported by the Great Project of Natural Science Foundation from Anhui

445

Provincial Education Department (ZD200910), the Natural Science Foundation of Anhui

16

446

Province (070411009), and the innovation funding to undergraduate students at HFUT

447

(XS08072, 0637).

448 449

References

450

Apel K, Hirt H (2004). Reactive oxygen species: metabolism, oxidative stress, and signal

451

transduction. Annu. Rev. Plant Boil. 55, 373−399.

452

Bartosz G (1997). Oxidative stress in plants. Acta Physiol. Plant 19, 47−64.

453

Bloem E, Riemenschneider A, Volker J, Papenbrock J, Schmidt A, Salac I et al. (2004).

454

Sulphur supply and infection with Pyrenopeziza brassica influence L-cysteine

455

desulfhydrase activity in Brassica napus L. J. Ex. Bot. 55, 2305−2312.

456 457 458 459

Blokhina O, Virolainen E, Fagerstedt KV (2003). Antioxidants, oxidative damage and oxygen deprivation stress: a review. Annal Bot. 91, 179−194. Bowler C, Fluhr R (2000). The role of calcium and activated oxygens as signals for controlling cross-tolerance. Trends Plant Sci. 5, 241−246.

460

Cao ZY, Xuan W, Liu ZY, Li XN, Zhao N, Xu P et al. (2007). Carbon monoxide

461

promotes lateral root formation in rapeseed. J. Integr. Plant Biol. 49, 1070−1079.

462

Deising H, Nicholson RL, Haug M, Howard RJ, Mendgen K (1992). Adhesion pad

463

formation and the lnvolvement of cutinase and esterases in the attachment of

464

uredospores to the host cuticle. Plant Cell 4, 1101−1111.

465 466

Delledonne M (2005). NO news is good news for plants. Curr. Opini. Plant Biol. 8, 390−396.

467

Foy CD (1984). Physiological effects of hydrogen, Al and manganese toxicities in acid soil.

468

In: Adams F. ed. Soil acidity and liming. American Society of Agronomy, Madison,

469

pp. 57−97.

470

García-Limones C, Hervás A, Navas-Cortés JA, Jiménez-Díaz RM, Tena M (2002).

471

Induction of an antioxidant enzyme system and other oxidative stress markers

472

associated with compatible and incompatible interactions between chickpea (Cicer

473

arietinum L.) and Fusarium oxysporum f. sp. ciceris. Physiol. Mol. Plant Pathol. 61,

17

474 475 476

325−337. Hällgren JE, Fredriksson SÅ (1982). Emission of hydrogen sulfide from sulfate dioxide-fumigated pine trees. Plant Physiol. 70, 456−459.

477

Han Y, Xuan W, Yu T, Fang WB, Lou TL, Gao Y et al. (2007). Exogenous hematin

478

alleviates mercury-induced oxidative damage in the roots of medicago sativa. J.

479

Integr. Plant Biol. 49, 1703−1713.

480 481

Horst WJ, Asher CJ, Cakmak I, Szulkiewicz P, Wissemeier AH (1992). Short-term responses of soybean roots to aluminium. J. Plant Physiol. 140, 174−178.

482

Hosoki R, Matsuki N, Kimura H (1997). The possibel role of hydrogen sulfide as an

483

endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem. Biophys.

484

Res. Commun. 237, 527−531.

485

Huang BK, Xu S, Xuan W, Li M, Cao ZY, Liu KL et al. (2006). Carbon monoxide

486

alleviates salt-induced oxidative damage in wheat seedling leaves. J. Integr. Plant

487

Biol. 48, 249–254.

488

Jones DL, Kochian LV (1995). Aluminium inhibition of the inositol 1,4,5-triphosphate

489

signal transduction pathway in wheat roots: a role in aluminium toxicity? Plant Cel1

490

7, 1931−1922.

491 492 493 494 495 496 497 498

Knight H, Knight MR (2001). Abiotic stress signaling pathways: specificity and cross-talk. Trends Plant Sci. 6, 262−267. Kuo MC, Kao CH (2003). Aluminum effects on lipid peroxidation and antioxidative enzyme activities in rice leaves. Biol. Plant. 46, 149–152. Li L, Bhatia M, Moore PK (2006). Hydrogen sulphide- a novel mediator of inflammation? Curr. Opin. Pharmacol. 6, 125–129. MacDonald T, Martin RB (1988). Aluminum ion in biological systems. Trends. Biochem. Sci. 13, 15−19.

499

Marschner H (1995). Mineral Nutrition of Higher Plants (Second Edition). Cambrige

500

Mittler R (2002). Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7,

501 502 503

405−410. Mittler R (2006). Abiotic stress, the field environment and stress combination. Trends Plant Sci. 11, 15–19.

18

504 505 506 507 508 509 510

Rausch T, Wachter A (2005). Sulfur metabolism, a versatile platform for launching defence operations. Trends Plant Sci. 10, 503−509. Rennenberg H (1983). Role of O-acetylserine in hydrogen sulfide emission from pumpkin leaves in response to sulfate. Plant Physiol. 73, 560−565. Rennenberg H (1984). The fate excess of sulfur in higher plants. Annu. Rev. Plant Physiol. 35, 121−153. Rennenberg H, Huber B, Schröder P, Stahl K, Haunold W, Georgii HW et al. (1990).

511

Emission of volatile sulfur compounds from spruce trees. Plant Physiol. 92, 560−564.

512

Riemenschneider A, Nikiforova V, Hoefgen R, De Kok LJ, Papenbrock J (2005a).

513

Impact of elevated H2S on metabolite levels, activity of enzymes and expression of

514

genes involved in cysteine metabolism. Plant Physiol. Biochem. 43, 473−483.

515

Riemenschneider A, Wegele R, Schmidt A, Papenbrock J (2005b). Isolation and

516

characterization of a D-cysteine desulfhydrase protein from Arabidopsis thaliana.

517

FEBS J. 272, 1291–1304.

518 519

Sekiya J, Schmidt A, Wilson LG, Filner P (1982a). Emission of hydrogen sulfide by leaf tissue in response to L-cysteine. Plant Physiol. 70, 430−436.

520

Sekiya J, Wilson LG, Filner P (1982b). Resistance to injury by sulfur dioxide, Correlation

521

with its reduction to, and emission of, hydrogen sulfide in cucurbitaceae. Plant

522

Physiol. 70, 437−441.

523

Shewry PR (1995). Plant storage protein. Bio. Rev. 70, 375−426.

524

Stuiver CEE, De Kok LJ, Kuiper PJC (1992). Freezing tolerance and biochemical

525

changes in wheat shoots as affected by H2S fumigation. Plant Physiol. Biochem. 30,

526

47–55.

527 528

Surrey K (1964). Spectrophotometric method for determination of lipoxidase activity. Plant Physiol. 39, 65−70.

529

Tahara K, Yamanoshita T, Norisada M, Hasegawa I, Kashima H, Sasaki S et al.

530

(2008). Aluminum distribution and reactive oxygen species accumulation in root tips

531

of two Melaleuca trees differing in aluminum resistance. Plant Soil 307, 167–178.

532

Van Breusegem F, Vranová E, Dat JF, Inze D (2001). The role of active oxygen species

19

533 534 535

in plant signal transduction. Plant Sci. 161, 405–414. Wang R (2002). Two’s company, there’s a crowd, can H2S be the third endogenous gaseous transmitter? FASEB J. 16, 1792−1798.

536

Wang YS, Wang J, Yang ZM, Wang QY, Lu B, Li SQ et al. (2004). Salicylic acid

537

modulates aluminum-induced oxidative stress in roots of Cassia tora. Acta Bot. Sin.

538

46, 819−828.

539

Wang YS, Yang ZM (2005). Nitric oxide reduces aluminum toxicity by preventing

540

oxidative stress in the roots of Cassia tora L. Plant Cell Physiol. 46, 1915–1923.

541

Wilson LG, Bressan RA, Filner P (1978). Light-dependent emission of hydrogen sulfide

542

from plants. Plant Physiol. 61, 184−189.

543

Winner WE, Smith CL, Koch GW, Mooney HA, Bewley JD, Krouse HR (1981). Rates

544

of emission of H2S from plants and patterns of stable sulfur isotope fractionation.

545

Nature 289, 672−673.

546

Xu S, Sa ZS, Cao ZY, Xuan W, Huang BK, Ling TF et al. (2006). Carbon monoxide

547

alleviates wheat seed germination inhibition and counteracts lipid peroxidation

548

mediated by salinity. J. Integr. Plant Biol. 48, 1168–1176.

549

Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K et al. (2008). H2S as a physiologic

550

vasorelaxant, hypertension in mice with deletion of cystathionine γ-lyase. Science 322,

551

587−590.

552

Zhang H, Shen WB, Zhang W, Xu LL (2005). A rapid response of β-amylase to nitric

553

oxide but not gibberellin in wheat seeds during the early stage of germination. Planta

554

220, 708–716.

555

Zhang H, Hu LY, Hu KD, He YD, Wang SH, Luo JP (2008a). Hydrogen sulfide

556

promotes wheat seed germination and alleviates the oxidative damage against copper

557

stress. J. Integr. Plant Biol. 50, 1518−1529.

558

Zhang H, Li YH, Hu LY, Wang SH, Zhang FQ, Hu KD (2008b). Effects of exogenous

559

nitric oxide donor on antioxidant metabolism in wheat leaves under aluminum stress.

560

Russ. J. Plant Physiol. 55, 469–474.

561

Zhang H, Tang J, Liu XP, Wang Y, Yu W, Peng WY, et al. (2009a). Hydrogen sulfide

20

562

promotes root organogenesis in Ipomoea batatas, Salix matsudana and Glycine max.

563

J. Integr. Plant Biol. 51, 1084–1092.

564

Zhang H, Ye YK, Wang SH, Luo JP, Tang J, Ma DF (2009b). Hydrogen sulfide

565

counteracts chlorophyll loss in sweetpotato seedling leaves and alleviates oxidative

566

damage against osmotic stress. Plant Growth Regul. 58, 243–250

567 568

Zheng SJ, Yang JL (2005). Target sites of aluminum phytotoxicity. Biol. Plant. 49, 321−331.

21

569

Tables

570

Table 1. Inhibitory effect of Al3+ on the germination of wheat seeds. Concentration 3+

of Al

(mmol/L) 0 5

Germination

Length of radicle

Length of

Radicle number

percentage (%)

(cm)

coleoptile (cm)

(50 seeds)

96.4±2.1

2.7±0.9

1.6±0.4

153±9.3

98.2±1.1

1.5±0.3 1.4±0.1 1.0±0.2 0.7±0.2 0.8±0.2

148±6.9 142±5.7 126±7.3 68±4.6 48±5.8

10

96.5±2.5

15 20

85±4.2 73.7±3.6

25

60.9±4.7

2.1±0.3 1.1±0.2 0.7±0.2 0.4±0.3 0.3±0.1

30 60

51.1±4.3 42.3±5.1

0.16±0.1 0.1±0.02

0.6±0.2 0.3±0.1

38±6.5 16±4.3

90 120 150

30.2±6.1 21.3±6.3 9.7±5.6

0 0 0

0.3±0.2 0.2±0.1 0.1±0.02

0 0 0

571

Wheat seeds were exposed to 0, 5, 10, 15, 20, 25, 30, 60, 90, 120, 150 mmol/L AlCl3 for 48

572

h. Values are the means±S.D. (n = 6).

22

573

Table 2. Effects of NaHS treatment on wheat seed germination under normal condition. Concentration of NaHS treatment (mmol/L) Germination percentage (%) Length

of

radicle (cm) Length of coleoptile (cm) Radicle number (50 seeds)

0.0

0.3

0.6

0.9

1.2

1.5

92.7±3.2 a

95.2±6.4 a

90.1±5.7 a

91.2±4.8 a

91.6±5.7 a

89±3.8 a

1.23±0.17a

1.32±0.24a

1.15±0.26a

1.33±0.41a

1.36±0.51a

1.18±0.33a

0.96±0.23a

0.98±0.17a

1.03±0.47a

0.92±0.25a

0.98±0.25a

1.07±0.23a

146.7±8.8a

153±6.7 a

149.6±4.8a

158.6±9.7 a

141.3±11.4a

158.3±7.1 a

574

Wheat seeds were cultured in 0, 0.3, 0.6, 0.9, 1.2, 1.5 mmol/L NaHS respectively for 36 h,

575

and then the germination percentage was investigated. Values are the means±S.D. (n = 6).

576

Different letters mean significance of difference between the treatments (P